identification of hypoxia -inducible target genes of aspergillus … · 1 identification of hypoxia...
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Identification of hypoxia-inducible target genes of Aspergillus fumigatus by 1
transcriptome analysis reveals cellular respiration as important contributor 2
to hypoxic survival 3
4
Kristin Krolla*
, Vera Pähtza,g*
, Falk Hillmanna,e
, Yakir Vakninf, Wolfgang Schmidt-Heck
b, 5
Martin Rothc, Ilse D. Jacobsen
d, Nir Osherov
f, Axel A. Brakhage
a,e and Olaf Kniemeyer
a,e,g # 6
7
aDepartment of Molecular and Applied Microbiology,
bResearch Group Systems Biology / 8
Bioinformatics, cBio Pilot Plant,
dResearch group Microbial Immunology, the Leibniz 9
Institute for Natural Product Research and Infection Biology – Hans Knöll Institute (HKI), 10
Jena, Germany, eDepartment of Microbiology and Molecular Biology, Institute of 11
Microbiology, Friedrich Schiller University Jena, Jena, Germany, fDepartment of Clinical 12
Microbiology and Immunology, Aspergillus and Antifungal Research Laboratory, Sackler 13
School of Medicine, Tel-Aviv University, Ramat-Aviv, Tel-Aviv, Israel, gIntegrated Research 14
Treatment-Center, Center for Sepsis Control and Care (CSCC), University Hospital Jena, 15
Germany 16
17
*Contributed equally to this work 18
# Address correspondence to Olaf Kniemeyer, [email protected] 19
20
Running title: Hypoxia induced respiratory genes in A. fumigatus 21
22
EC Accepts, published online ahead of print on 1 August 2014Eukaryotic Cell doi:10.1128/EC.00084-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Abstract (222 words) 23
Aspergillus fumigatus is an opportunistic, airborne pathogen causing invasive aspergillosis in 24
immunocompromised patients. During the infection process A. fumigatus is challenged by 25
hypoxic microenvironments occurring in inflammatory, necrotic tissue. To gain further 26
insights into the adaptation mechanism, A. fumigatus was cultivated in an oxygen-controlled 27
chemostat under hypoxic and normoxic conditions. Transcriptome analysis revealed a 28
significant increase of transcripts associated with cell wall polysaccharide metabolism, amino 29
acid and metal ion transport, nitrogen metabolism and glycolysis. A concomitant reduction in 30
transcript levels was observed with cellular trafficking and G-protein coupled signaling. To 31
learn more about the functional roles of hypoxia-induced transcripts we deleted A. fumigatus 32
genes putatively involved in reactive nitrogen species detoxification (fhpA), NAD+ 33
regeneration (frdA, osmA) nitrogen metabolism (niaD, niiA) and respiration (rcfB). We show 34
that the NO-detoxifying flavohemoprotein gene fhpA is strongly induced by hypoxia 35
independent of the nitrogen source, but is dispensable for hypoxic survival. By deleting the 36
nitrate reductase gene niaD, the nitrite reductase gene niiA and the two fumarate reductase 37
genes frdA and osmA, we found that alternative electron acceptors such as nitrate and 38
fumarate do not have a significant impact on growth of A. fumigatus during hypoxia, but that 39
functional mitochondrial respiratory chain complexes are essential under these conditions. 40
Inhibition studies indicated that primarily complex III and IV play a crucial role in the 41
hypoxic growth of A. fumigatus. 42
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Introduction 44
Aspergillus fumigatus is a ubiquitously distributed saprophytic fungus mainly found in the 45
soil where it plays an essential role in the degradation of organic debris (1, 2). The fungus 46
propagates by formation of conidia, which easily disperse into the air. During daily life, 47
humans inhale hundreds of A. fumigatus conidia, which are efficiently eliminated by the host 48
immune system (3, 4). However, in immunocompromised patients, conidia are able to 49
germinate and grow, eventually leading to an invasive infection called invasive aspergillosis 50
(IA). Due to a rising number of immunodeficient patients, the number of cases of invasive 51
aspergillosis has increased in the last decades. The diagnosis of IA is still difficult and thus, 52
mortality rates are unacceptably high ranging from 50-90 % (5). In addition to these clinical 53
aspects of this disease, the pathophysiology of A. fumigatus infections is insufficiently 54
understood. To date, no classic virulence factor has been identified for A. fumigatus and 55
consequently the adaptation mechanisms of A. fumigatus towards the host environment have 56
become a main focus of research. 57
During infection, long-term low oxygen conditions challenge this obligate aerobe fungus: O2 58
levels drop from 21 % in the atmosphere to 14 % in the lung alveoli (6). In the surrounding 59
tissue, oxygen availability is further reduced to 2-4 % and in inflammatory tissue to even less 60
than 1 % (7). Such growth conditions were proposed to shape the pathobiology of A. 61
fumigatus, and their in vivo relevance was further supported by a hypoxia-sensitive mutant 62
(ΔsrbA), which was avirulent in a mouse model of invasive aspergillosis (8, 9). However, the 63
metabolic response of A. fumigatus to hypoxia is less well understood (10). The role of 64
fermentation during hypoxic growth remains ambiguous and the electron transport chain 65
(ETC) is still active at low oxygen levels for energy production (11). This was also shown in a 66
previous report on hypoxia-induced changes of the transcriptome and proteome in A. 67
fumigatus over a period of 24 h (here defined as short-term response) (12). In this work, the 68
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authors used a batch-cultivation and monitored the A. fumigatus hypoxic response at the time 69
points 2, 6, 12 and 24 hours. In a recent proteomic study of our group on the long-term 70
response (5-6 days) towards hypoxia A. fumigatus was cultivated in an oxygen-controlled 71
chemostat. Under these conditions an increased abundance of all mitochondrial respiratory 72
chain complexes, especially complex III and IV was observed (13). In the same study, 73
activation of NO-detoxifying enzymes was detected. There is some indication that complex 74
IV may produce NO under oxygen-limiting conditions as shown in baker`s yeast (14). The 75
increased levels of NO cause post-translational modifications of proteins, e.g. 76
nitrotyrosination, which may lead to the induction of hypoxic genes (15). Hence, it is 77
supposed that the mitochondrial electron transport chain plays a role in oxygen sensing and 78
hypoxic signaling (16, 17). 79
However, when the terminal electron acceptor oxygen is limited, many organisms switch 80
from respiration to fermentation to regenerate NAD+ and to maintain ATP synthesis. Several 81
examples for such alternative electron sinks have already been described in diverse fungal 82
species. In A. oryzae and A. nidulans, genes involved in ethanol fermentation are induced 83
during hypoxic growth conditions (18). However, for A. fumigatus no growth phenotype was 84
observed under oxygen limiting conditions for strains devoid of ethanol fermentation (19). 85
The GABA-shunt pathway has also been proposed to bypass the accumulation of NADH in A. 86
fumigatus and A. nidulans during hypoxic growth (12, 20, 21). Furthermore, in A. nidulans 87
and Fusarium oxysporum, growth under oxygen-limiting conditions is supported by using 88
nitrate as an alternative electron acceptor, a process also referred to as ammonia fermentation 89
(22, 23). Taken together, filamentous fungi posses a broad range of metabolic pathways to 90
maintain energy levels under oxygen limiting conditions, but so far their physiological impact 91
on A. fumigatus during hypoxia has remained largely unexplored. 92
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In our previous, aforementioned proteomic study we established an oxygen-controlled, 93
glucose-limited chemostat as a model for highly reproducible hypoxic cultivations of A. 94
fumigatus (13). A following up proteomic study on the short-term response of A. fumigatus to 95
hypoxia (2-24 hours) revealed a surprisingly low correlation between the proteomic data of 96
the two studies (12). This may be explained by the different incubation times (6 days versus 97
2-24 hours) or culture conditions (glucose-limited chemostat versus batch culture without 98
glucose limitation). To provide further insights into the long-term adaptation mechanism of A. 99
fumigatus towards hypoxia we cultivated A. fumigatus in either normoxic (21 % pO2) or 100
hypoxic (0.21 % pO2) conditions as described in Vödisch et al. (13) and compared the 101
underlying changes of the transcriptome by microarray analysis. Gene expression at the 102
mRNA level correlated well with our previous proteome data (13), but also suggested an 103
important role for alternative NAD+-regenerating systems and hypoxic NO signaling. 104
Inhibiting the underlying pathways by gene deletion revealed that alternative electron 105
acceptors such as nitrate and fumarate do not have a significant impact on growth during 106
hypoxia in A. fumigatus, whereas functional mitochondrial respiratory chain complexes are 107
essential under these conditions. 108
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Methods 110
111
Strain and culture conditions 112
A. fumigatus was grown in Aspergillus minimal medium (AMM) as previously described (24) 113
containing 60 mM glucose and 70 mM NaNO3 as sole carbon and nitrogen source, 114
respectively. To study the long-term response of A. fumigatus to hypoxic growth conditions, 115
A. fumigatus strain ATCC 46645 was grown in a glucose-limited oxystat (chemostat with 116
constant oxygen partial pressure) in a continuous culture as described previously by Vödisch 117
et al., 2011 (13). Batch fermentation was used to analyze the short-term response of A. 118
fumigatus to hypoxic growth conditions. Fermentation was carried out as described previously 119
(12). For expression analysis of the flavohemoprotein fhpA either 70 mM NaNO3 or 20 mM 120
L-glutamine were used as nitrogen sources in batch cultivations of A. fumigatus. All strains 121
used in this study are listed in supplementary table S1. 122
123
RNA preparation and transcriptome analysis 124
For microarray analyses, total RNA was isolated from three independent chemostat cultures, 125
grown either under normoxic or hypoxic conditions. Frozen mycelium of A. fumigatus ATCC 126
46645 was ground to a fine powder and 100 mg were used for total RNA isolation using the 127
Qiagen “RNeasy Mini Kit” according to the manufacturer’s instructions. Following DNase 128
treatment (TurboDNA-free kit, Ambion, Germany) the quantity and quality of RNA 129
preparations were determined spectrophotometrically with a nano-drop (Thermo Fisher 130
Scientific, Germany). 131
Full genome transcriptomic analyses were performed at Febit (Heidelberg, Germany) as 132
described previously in Gehrke et al., 2010 (25). Briefly, custom made Geniom biochips®
133
comprising ~15.000 oligonucleotide probes were designed, covering the expected number of 134
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transcripts based on previous genomic sequence data (26). All data analyses of the FEBIT 135
microarrays were performed using ‘LIMMA’ (Linear Models for Microarray Data) packages 136
(27) of the Bioconductor Software (28). Background correction was performed using the 137
intensities of blank probes which consist of only one single ”T” nucleotide. The median 138
background intensity is subtracted from spot intensity. After converting any negative values to 139
low positive value, signal intensities were log2-transformed, and duplicate spots were 140
averaged. The obtained data were processed using quantile normalization. To obtain the genes 141
with the most evidence of differential expression, a linear model fit was applied for each gene 142
using LIMMA. Candidate genes for further analysis were selected on the basis of their fold-143
change ( 2) and p-value ( 0.05). Data of the microarray analyses have been deposited in the 144
OmniFung Data Warehouse [http://www.omnifung.hki-jena.de] and ArrayExpress (acc-145
number E-MTAB-2699). These data can be easily accessed by public login. 146
147
Nucleic acid hybridizations 148
Gene deletions were verified by Southern hybidizations. Briefly, genomic DNA of A. 149
fumigatus was extracted using the MasterPure yeast DNA purification kit (Epicentre 150
Biotechnologies, USA) and digested by specific restriction enzymes (New England Biolabs, 151
Germany). Resulting DNA fragments were separated by agarose gel (1 % w/v) 152
electrophoresis and transferred onto HybondTM
-N+ membranes (GE Healthcare Bio-Sciences, 153
Germany) by capillary blotting. Gene specific DNA probes were generated by PCR including 154
digoxigenin labeled dUTPs (Jena Bioscience, Germany) or [α-32
P]dCTP (Institute of Isotopes 155
C. Ltd, Hungary). DIG-labeled probes were hybridized with DIG Easy Hyb and detected 156
using Anti-digoxigenin antibodies and CDP-Star as an ultrasensitive fluorescent substrate of 157
alkaline phosphatase (Roche Applied Science, Germany). Oligonucleotides used for 158
amplifying labeled DNA probes are shown in supplementary table S2. For Northern 159
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hybridizations 10 µg of total RNA were separated on a denaturing agarose gel (1.2 % (w/v) 160
agarose, 40 mM MOPS, 10 mM sodium acetate, 2 mM EDTA, 2 % (v/v) formaldehyde, pH 161
7), Blotting, hybridzation and detection were done as described above. Primers used to 162
generate mRNA specific DNA-probes are listed in supplementary table S3. 163
164
Generation of fungal strains 165
Mutants of A. fumigatus CEA17ΔakuBKU80
(29) were generated by homologous 166
recombination following the transformation of protoplasts (30). All mutant strains used in this 167
study are listed in supplementary table S1. 168
For deletion of the fumarate reductase frdA (AFUA_7G05070) the flanking regions were 169
amplified from genomic DNA with the primer pairs FrdA_LF_fw/FrdA_LFhph_rev and 170
FrdA_RFhph_fw/FrdA_RF_rev. This reaction produces overlapping ends to the hygromycin 171
resistance cassette at the 3'-end of the upstream flanking region and at the 5'-end of the 172
downstream flanking region of the frdA gene. The hygromycin resistance cassette was 173
amplified from plasmid pUChph (31) using the primers Hph_fw and Hph_rev. The frdA 174
deletion construct was obtained by a 3-fragment PCR using the primers FrdA_LF_fw and 175
FrdA_RF_rev. Similary, the SREBP transcription factor srbA (AFUA_2G01260) was deleted 176
by amplifying the corresponding flanking region using the primers 177
SrbA_LF_fw/SrbA_LFhph_rev and SrbA_RFhph_fw/SrbA_RF_rev. 178
For deletion of the fumarate reductase osmA (AFUA_8G05530), the nitrate reductase niaD 179
(AFUA_1G12830) and the nitrite reductase niiA (AFUA_1G12840) genes, the corresponding 180
flanking regions were amplified using the primer pairs OsmA_LF_fw/OsmA_LFptrA_rev and 181
OsmA_RFptrA_fw/OsmA_RF_rev, NiaD_LF-fw/NiaD_LFptrA_rev and NiaD_RFptrA-182
fw/NiaD_RF_rev, and NiiA_LF-fw/NiiA_LFptrA_rev and NiiA_RFptrA-fw/NiiA_RF_rev, 183
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respectively. Here, pyrithiamine resistance was used as selection marker and the respective 184
gene was amplified from plasmid pSK275 (32) using the primers PtrA_fw and PtrA_rev. 185
For the generation of the double knock-out strain ΔfrdA/ΔosmA the osmA deletion cassette 186
containing pyrithiamine resistance (as described above) was used to transform the ΔfrdA 187
strain. Transformants were selected on AMM plates containing either 0.1 µg/mL pyrithiamine 188
(Sigma, Germany) or 240 µg/mL hygromycin (Roche Applied Science, Germany). 189
To generate a C-terminal eGFP fusion protein of the flavohemoprotein FhpA (AFUA_ 190
4G03410), 1 kb of the native promoter and the fhpA gene were amplified from genomic DNA 191
of A. fumigatus CEA17ΔakuBKU80
using the primers FhpA_GFP_HindIII_ and 192
FhpA_GFP_BamHI_rev. The obtained DNA fragment was inserted into the vector 193
p123_eGFP (33) containing the hygromycin resistance cassette. The resulting vector 194
pfhpA_egfp was ectopically integrated into the genome of A. fumigatus CEA17ΔakuBKU80
195
∆fhpA (derived from M. Vödisch, unpublished data). 196
All PCR reactions were performed with Phusion High-Fidelity DNA Polymerase (Thermo 197
Fisher Scientific, Germany) according to the manufacturer's recommendations. All 198
oligonucleotides used for the generation of mutant strains are listed in supplementary table 199
S2. 200
The deletion construct for rcfB (AFUA_1G12250) was prepared using the yeast 201
recombinational cloning technique (34). Primers were designed and synthesized with the 5’ 202
common regions highlighted in italic in supplementary table S2. The hygromycin cassette 203
(Hph), containing 5′ and 3′ HindIII and XbaI restriction sites, was generated by PCR 204
amplification using primers Hph 5′ and Hph 3′. Yeast shuttle vector pYes2 was digested with 205
HindIII and XbaI. Yeast strain BY4741 was used for yeast transformation following the 206
protocol described by Collopy et al., 2010 (34). Yeast DNA was prepared with the 207
Masterpure Yeast DNA Purification Kit (Epicentre Biotechnologies, USA). Transformation of 208
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A. fumigatus CEA17ΔakuBKU80
protoplasts with linearized plasmids ΔAfu1g12250-Hph 209
(EcoRV) was carried out as previously described (35). 210
211
Growth susceptibility assays 212
Conidia were harvested after 5 days of cultivation on AMM agar plates with sterile water and 213
filtered through a 40-μm cell strainer (BD Biosciences, Germany). Conidia were serially 214
diluted in sterile water to obtain defined concentrations and 105, 10
4, 10
3 and 10
2 conidia were 215
spotted in a volume of 5 µL on AMM agar plates in the presence of certain stress-inducing 216
agents. Growth was documented after 48 or 72 h incubation at 37 °C. 217
To generate nitrosative stress conditions, different concentrations of sodium nitrite were 218
added in acidified AMM (pH 4.5). Reductive stress conditions were induced by the addition 219
of 10 mM TCEP-HCL (final concentration). 220
Normoxic or two hypoxic atmospheres consisting of either 1 % O2 and 5 % CO2 (HeraCell 221
150, Thermo Fisher Scientific, Germany) or 0.2 % O2 and 5 % CO2 (H35 Hypoxystation, Don 222
Whitley Scientific, UK) were used to monitor growth of A. fumigatus on AMM agar plates. 223
A. fumigatus wild-type strain and the hypoxia sensitive strain srbA were cultivated in the 224
presence of specific respiratory chain inhibitors (Sigma Aldrich, Germany): 1, 2, 3, and 4 mM 225
rotenone (complex I inhibitor); 50, 75, 100 and 150 µM flavone (inhibitor of alternative 226
NADH:ubiquinone oxidoreductases); 100, 200, 300, 400 and 500 mM disodium malonate 227
(complex II inhibitor); 5, 10 and 20 µM antimycin A (complex III inhibitor); 0.25, 0.5 and 1 228
mM sodium azide (complex IV inhibitor); 5, 10 and 20 µM oligomycin (complex V 229
inhibitor); 0.5, 1 and 2 mM 2,4-dinitrophenole (transmembrane H+ carrier inhibitor) and 2.5, 5 230
and 10 mM salicylhydrosamic acid (alternative oxidase inhibitor). 5 µL aliquots of each strain 231
were spotted in 10-fold serial dilutions on AMM agar plates containing the respective 232
inhibitor at the concentration as indicated above. 233
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Reductive stress assay 234
To monitor growth in presence of the reductive stress agent TCEP-HCl (Merck KGaA, 235
Germany) in liquid culture (96 MTP format), 2,000 conidia were inoculated per well, 236
containing AMM supplemented with 0-20 mM TCEP-HCl. The plate was incubated for 3 237
days at 37 °C and growth was documented. 238
239
Determination of succinic acid concentration 240
The concentration of succinic acid in the culture supernatant was quantified by an enzymatic 241
detection kit according to the manufacturer`s instructions (UV-test for succinic acid, R-242
Biopharm, Germany). 243
244
Fluorescent microscopy studies 245
For fluorescence imaging, 50 µL AMM (containing either nitrate [70 mM] or glutamine [20 246
mM] as nitrogen source) were inoculated with 1 x 104 conidia of an FhpA_eGFP strain on 247
glass cover slips in a wet chamber. After 14 h precultivation under normoxic conditions at 37 248
°C samples were shifted to a hypoxic atmosphere with 1 % O2 and 5 % CO2 at 37 °C 249
(HeraCell 150, Thermo Fisher Scientific, Germany). Samples were analyzed after 0, 3, 6, 12 250
and 24 h of hypoxia using a Leica DMI 4000B fluorescence microscope (Leica Microsystems, 251
Germany). Images were taken with a Leica DFC480 camera and analyzed by Leica LAS 252
V.3.7 software. 253
254
Egg infection model 255
Virulence of A. fumigatus CEA17ΔakuBKU80
and the deletion strains ∆frdA, ∆osmA, 256
∆frdA/∆osmA and ∆niiA was tested in an established egg model for Aspergillus species (36). 257
Briefly, after 7 days cultivation on Malt agar plates at room temperature, conidia were 258
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harvested in PBS containing 0.1 % Tween 20 (v/v) and filtered with a 40 µM cell strainer (BD 259
Bioscience, Germany). After counting conidia using either a counting chamber (Roth, 260
Germany) or the CASY®
Cell counter analyzer TT (Roche Applied Science, Germany) 261
conidia were diluted with PBS to a concentration of 104 conidia/mL just prior infection. PBS 262
alone was used as a negative control. Embryonated eggs were incubated at 37.6 °C and 50-60 263
% relative humidity (BSS 300, Grumbach, Germany). After 10 days of incubation twenty 264
eggs per group were infected with 103 conidia/egg via the chorioallantoic membrane (CAM) 265
and survival was monitored daily over 7 days by candling. Survival data were plotted as 266
Kaplan-Meyer curves and statistically analyzed by a log rank test using Graph Pad Prism 5.00 267
(GraphPad Software, USA). 268
269
Statistics 270
The Student’s t-test was used for significance testing of two groups. Differences between the 271
groups were considered significant if p ≤ 0.05 or p ≤ 0.01. 272
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Results 274
275
Gene expression in long-term hypoxia adapted A. fumigatus 276
To study the effect of long-term exposure to hypoxia on the transcriptional profile of A. 277
fumigatus we cultivated the fungus in a chemostat, which allowed us to vary the oxygen 278
partial pressure (21 % pO2 and 0.21 % pO2), while all other cultivation parameters such as 279
glucose concentration, pH and temperature were kept constant. A dilution rate of 0.08 h-1
was 280
applied to ensure the same growth rate under normoxic and hypoxic conditions. Steady-state 281
growth was reached after between 3-4 days and the biomass of 750 mg dry weight l-1
was 282
harvested after 5-6 days followed by RNA extraction. 283
Febit Geniom Biochips representing 15,000 open reading frames were hybridized with RNA 284
from three replicates of each sample. We applied a principle component analysis method 285
(Sammon’s Non Linear Mapping) to determine sample variance and to identify outliers. One 286
sample outlier (0.21 % O2) was detected, which was excluded from further analysis. Genes 287
showing at least 2-fold changes in expression (p value ≤ 0.05) were considered to be 288
differentially expressed. 289
In total, 1,614 genes were up- and 1,260 genes were significantly down-regulated in A. 290
fumigatus during growth under hypoxia in a glucose-limited chemostat (data set S1). Gene set 291
enrichment (GSE) analysis based on FunCat and GO classification was performed to gain a 292
general overview of the type of categories affected by hypoxia (Fig. 1 and Fig. S1). Specific 293
categories of transcripts that were significantly up-regulated under hypoxia were associated 294
with nuclear and RNA-transport, energy conversion, polysaccharide degradation, DNA 295
topology, enzyme activation, amino acid-, metal ion- and transmembrane transport, nitrogen 296
compound and metabolic process, the glyoxylate cycle, gluconeogenesis and methionine 297
biosynthesis. Furthermore, several flavine-containing enzymes involved in oxidation-298
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reduction processes were also up-regulated. As expected, many additional metabolic changes 299
occurred in response to hypoxia. Many glycolytic gene transcripts and several transcripts 300
related to ethanol fermentation showed higher mRNA abundance in hypoxia: a pyruvate 301
decarboxylase (AFUA_3G11070) along with two alcohol dehydrogenases, alcC 302
(AFUA_5G06240) and adh2 (AFUA_2G10960). In agreement with previous findings from 303
other fungi that the GABA shunt could reduce NADH accumulation at low oxygen levels, we 304
found transcription of the succinate-semialdehyde dehydrogenase gene (AFUA_3G07150) to 305
be significantly higher in response to hypoxia. Considering transcription factors, transcript 306
levels of the central regulator of hypoxia adaptation, SrbA (AFUA_2G01260), was also 307
activated as the mRNA of this transcription factor was increased 4.9-fold, which in turn 308
activated key enzymes of sterol metabolism. An increased need for iron under hypoxia is 309
illustrated by the fact that the iron regulator HapX (AFUA_5G03920) was slightly up-310
regulated (1.2-fold), whereas the repressor of iron acquisition, SreA (AFUA_5G11260), 311
showed lower transcript levels (-2.8) under hypoxia. 312
Other FunCat categories were significantly reduced in response to hypoxia, and include 313
amongst others cytoskeleton/structural proteins, ER to Golgi transport, modification by 314
phosphorylation, osmosensing and response, cell redox homeostasis, phospholipid 315
metabolism and G-protein mediated transduction. Presumably, in hypoxic grown-cells there is 316
a reduction in cellular trafficking and signaling activity via G-protein coupled receptors and 317
small GTPases. 318
When comparing these gene expression data to the hypoxia proteome from our previous study 319
(13), we found a good correlation. Two thirds of the proteins (44 out of 66) showed the same 320
tendency of regulation as their corresponding transcript (Tab. S4). An opposite change in 321
abundance was observed for a few glycolytic enzymes, antioxidative proteins, enzymes of the 322
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TCA cycle and the electron transport chain, which suggests a regulation of these proteins at 323
the posttranscriptional level. 324
A comparison with the transcriptome data of the hypoxic response of A. fumigatus cultivated 325
in a batch-fermenter at low oxygen levels for 24 hours by Barker et al. (12) revealed that only 326
27% of the 867 significantly altered genes were also differentially expressed in our study (Fig. 327
S2). These transcripts included genes involved in metabolic processes such as amino acid, 328
lipid and terpenoid biosynthesis, cofactor and nucleotide metabolism, as well as replication, 329
cellular transport processes and protein folding/modifications. The biggest differences were 330
seen in the FunCat categories transcription and protein synthesis. Transcription levels of 331
genes involved in these processes decreased only significantly in the study of Barker et al. 332
(12). This suggests that transcription and translation is only transiently down-regulated during 333
hypoxia. 334
335
Confirmation of microarray data by Northern blot analyses 336
To confirm the robustness and quality of our transcriptome data from A. fumigatus cultivated 337
in an oxygen-controlled chemostat, we analyzed the gene expression of five selected genes 338
during hypoxia by Northern hybridization (Fig. 2). These included genes involved in reactive 339
nitrogen species detoxification (fhpA, AFUA_4G03410), NAD+ regeneration (frdA, 340
AFUA_7G05070), nitrogen metabolism (niiA, AFUA_1G12840) and respiration (cox5b, 341
AFUA_2G03010; rcfB, AFUA_1G12250). Hereby, RNA samples produced for our 342
microarray experiment were used. 343
The microarray data revealed a 17-fold up-regulation of the flavohemoprotein encoding gene 344
fhpA, which was also confirmed by Northern blot analysis (Fig. 2). This result is consistent 345
with the proteome analysis of both long- and short-term response of A. fumigatus under 346
hypoxic growth conditions (12, 13). Another interesting finding of the microarray study was 347
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the drastically increased expression (321-fold up-regulation) of the cytosolic fumarate 348
reductase gene frdA, which was also verified by Northern hybridizations (Fig. 2). For the gene 349
osmA, which encodes for the fumarate reductase isoenzyme OsmA (AFUA_8G05530), the 350
extent of induction was by more than one order of magnitude lower. However, Northern blot 351
analysis revealed no clear difference in the expression level of osmA during normoxia and 352
hypoxia (Fig. 2). 353
In addition, the microarray data revealed, that several transcripts linked to nitrate metabolism 354
were significantly increased (Tab. 1). Among them, the nitrite reductase niiA transcript 355
showed the highest increase in expression level. However, Northern blot analyses revealed 356
only a slight increase of niiA mRNA steady-state levels and no altered expression of nitrate 357
reductase niaD during hypoxic growth conditions (Fig. 2). 358
Interestingly, two components of the cytochrome c-oxidase respiratory complex, which acts 359
as the terminal enzyme of the respiratory chain, were also expressed at significantly higher 360
levels during hypoxia. This was subsequently confirmed by Northern hybridizations as well 361
(Fig. 2). These two genes encode the integral subunit CoxVb and an Rcf2-like protein, here 362
named RcfB. The orthologous gene from S. cerevisiae was also reported to be highly up-363
regulated under hypoxia (37, 38). The other known respiratory supercomplex factor similar to 364
RcfB, RcfA (AfuA_4G08130) was not significantly up-regulated under our experimental 365
hypoxic conditions. 366
Taken together, these data prompted us to analyze the impact of NO-detoxification, NAD+-367
regeneration and respiration on the hypoxic adaptation of A. fumigatus in more detail. 368
369
Nitrate-independent induction of the flavohemoprotein FhpA during hypoxia 370
To elucidate the role of the flavohemoprotein FhpA during hypoxia we characterized its 371
regulation in more detail. In Aspergilli, the NO dioxygenase activity of flavohemoproteins 372
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suggests a role in the detoxification of nitric oxide (NO), which is formed during nitrate 373
assimilation (39, 40). In agreement, it was shown that fhpA is expressed in the presence of 374
nitrate, but repressed when glutamine is used as sole nitrogen source (K. Lapp, M. Vödisch, 375
K. Kroll, V. Pähtz, S. Bruns, M. Strassburger, J. Linde, R. Guthke, O. Kniemeyer, V. 376
Valiante, T. Heinekamp and A. A. Brakhage, own unpublished data). This raises the question 377
whether the observed up-regulation of fhpA was induced by hypoxia or by the nitrate 378
assimilation process. Thus, A. fumigatus was cultivated after 14 h normoxic pre-cultivation 379
under hypoxic batch fermentation for 24 h, using either nitrate or glutamine as a sole nitrogen 380
source. Subsequently the expression of fhpA was monitored over time (Fig. 3A). In the 381
presence of nitrate, the hypoxia-induced expression of fhpA remained constant over time. In 382
contrast, when using glutamine as a nitrogen source the expression levels of fhpA increased 383
after 3 h of hypoxia, but dropped to their original level after longer periods of oxygen 384
depletion. Therefore, we conclude that nitrate promotes a stronger and longer-lasting 385
induction of fhpA expression under hypoxia than glutamine. These results were further 386
confirmed by expression analysis of a FhpA-eGFP fusion protein in an A. fumigatus strain 387
cultivated under similar conditions (Fig. 3B, Fig. S3). This suggests that the expression of the 388
flavohemoprotein fhpA is induced by hypoxia independently of the nitrogen source, whereas 389
the duration of expression is dependending on it. The contribution of FhpA to the hypoxic 390
survival of A. fumigatus was tested by cultivating an fhpA deletion (unpublished data) strain 391
under hypoxic (0.2 % O2) and normoxic (21 % O2) conditions with either nitrate or glutamine 392
as nitrogen source. However, the ∆fhpA strain showed a wild-type growth phenotype under 393
hypoxic conditions. 394
395
Nitrate metabolism during hypoxia 396
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Under anoxic conditions, a variety of bacteria and fungi such as Bacillus subtilis, Escherichia 397
coli or F. oxysporum are able to use alternative terminal electron acceptors (41-45). Among 398
them, nitrate (NO3-/NO2) with a relatively high redox potential, (E
’0 = +0.42 V) is the most 399
efficient electron acceptor besides oxygen (E’0
= +0.82 V). 400
However, no dissimilatory nitrate reduction has been reported for A. fumigatus. The fungus 401
assimilates nitrate via nitrite (NO2-) reduction to ammonium (NH4
+). These reactions are 402
catalyzed by the NADPH-dependent assimilatory nitrate and nitrite reductases, NiaD 403
(AFUA_1G12830) and NiiA (AFUA_1G12840), respectively. Whether this pathway may 404
serve as alternative electron sink during hypoxia in A. fumigatus has not been shown yet. 405
To study the impact of these two enzymes on the adaptation to hypoxia, we generated the 406
corresponding deletion mutants ∆niaD and ∆niiA (Fig. S4A-B). As expected, lack of niaD or 407
niiA abolished growth when nitrate was used as sole nitrogen source (data not shown). 408
Surprisingly, in the presence of other primary nitrogen sources, such as glutamine or 409
ammonium tartrate, growth of strain ∆niiA was also slightly reduced compared to the wild 410
type and the ∆niaD strain (Fig. S4C). However, no altered growth phenotype of the mutants 411
was observed under hypoxic conditions (Fig. S4C), which confirmed that these assimilatory 412
enzymes are not linked to respiratory nitrate ammonification in A. fumigatus during hypoxic 413
growth conditions. More likely, these enzymes may be involved in nitric oxide intermediate 414
(RNI) detoxification. It has been shown, that the mitochondrial cytochrome c oxidase Cco can 415
produce nitric oxide NO by reducing NO2
- under oxygen limiting conditions (14, 46-49). To 416
test whether the increased expression of niaD and niiA were caused by NO-formation during 417
hypoxia, ∆niaD, ∆niiA and the corresponding wild-type strain CEA17ΔakuBKU80
were 418
cultivated in the presence of sodium nitrite, which decomposes spontaneously to nitric oxide 419
at an acidic pH of 4.5 (50) (Fig. 4). Resistance towards NO of the ∆niaD-strain was similar to 420
the wild type, whereas growth of the ∆niiA-strain was visibly impaired (Fig. 4). However, 421
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agar diffusion assays with the NO-donor DETA-NO (data not shown) revealed no difference 422
in sensitivity between ∆niiA and the wild type. Taken together, these data suggest that the 423
nitrite reductase NiiA is only indirectly involved in the adaptation to hypoxic growth 424
conditions. Its role in detoxifying harmful RNIs during hypoxia remains unclear. Its slight up-425
regulation under these conditions may also simply be explained by a co-regulation with FhpA 426
as reported from A. nidulans (40). 427
To analyze the impact of niiA on virulence, embryonated eggs were infected with spores of 428
the ∆niiA strain. However, no significant differences in survival were observed between 429
embryonated eggs infected with the mutant and the corresponding wild-type strain (Fig. S5A). 430
431
Fumarate metabolism during hypoxia 432
Besides nitrate, fumarate represents another alternative electron acceptor in the absence of 433
oxygen. The yeast S. cerevisiae genome contains two soluble fumarate reductases that are 434
essential for anaerobic growth (51). In A. fumigatus, the cytosolic FAD dependent 435
oxidoreductase FrdA (AFUA_7G05070) revealed 43 % amino acid identity to the cytosolic 436
fumarate reductase Frd1 in S. cerevisiae. The second fumarate reductase of A. fumigatus 437
OsmA (AFUA_8G05530) was 42 % identical to the corresponding yeast homologue which is 438
localized in the promitochondria (51). Remarkably, under hypoxic conditions, fumarate 439
reductase frdA represented the gene with the highest induction found in our microarray study. 440
This is in agreement with the increased abundance of FrdA in the proteome of the short-term 441
response of A. fumigatus against hypoxia (12). 442
Next, we examined the time-dependent change of gene expression levels of fumarate 443
reductases frdA and osmA in more detail during a 24 h period of hypoxia (batch cultivation). 444
Similar to the results of the chemostat, during batch cultivation expression of frdA was 445
induced over all time points tested (Fig. 5A). In contrast, transcript levels of osmA were 446
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transiently increased within the first 3 h of hypoxia (Fig. 5A). When we quantified the 447
fumarate reductase product succinic acid we could indeed detect increased concentrations of 448
succinic acid in the culture supernatants of hypoxic batch fermentations. However, during 449
hypoxia the amount of succinic acid increased only slightly over time from 0.04 mM to 0.085 450
mM and the concentration remained low (Fig. 5B). 451
To investigate the role of both fumarate reductases during hypoxia in more detail frdA and 452
osmA were deleted in A. fumigatus (Fig. S6A-B) and the growth phenotypes of the obtained 453
knock-out strains frdA, osmA and the double mutant frdA/osmA were further 454
characterized. However, no significant differences were observed in growth between wild 455
type and knock-out strains under hypoxia (Fig. S6C-D). In yeast, fumarate reductases are 456
involved in the re-oxidation of FAD-prosthetic groups of flavoenzymes during anaerobiosis 457
(52). This fits with a slight growth defect of the frdA, osmA and frdA/osmA strains in the 458
presence of tris(2-chloroethyl)phosphate (TCEP) (Fig. 5C-D). TCEP reduces disulfide bonds 459
as sufficiently as DTT, but in contrast to DTT it is relatively resistant to air oxidation. All 460
three mutants did not grow in the presence of 20 mM TCEP in liquid AMM medium, whereas 461
the wild type still formed hyphae. Reducing agents as TCEP induce reductive stress and the 462
unfolded protein response (UPR) in fungal cells. Finally, to analyze any potential impact of 463
fumarate reductases on the virulence of A. fumigatus, the frdA, osmA and frdA/osmA 464
strains were tested in an embryonated egg infection model. However, no significant 465
differences in virulence between eggs infected with fumarate reductase deletion strains and 466
eggs infected with the wild type were observed, indicating that the enzymes are dispensable 467
for infection in this particular, alternative model (Fig. S5B). 468
Taken together, these data demonstrate that fumarate reductases are unlikely to function as 469
respiratory enzymes, but may contribute to regenerate the FAD/FMN prosthetic group of 470
flavoenzymes in A. fumigatus. 471
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472
Role of respiratory complexes during hypoxic adaptation 473
Despite previous indications for fermentation pathways, mitochondrial respiration has proven 474
to be essential during hypoxia (11, 13). To further investigate the robustness of this pathway 475
and the specific contribution of each individual respiratory complex to hypoxic survival, A. 476
fumigatus wild-type strain and the hypoxia sensitive strain srbA were cultivated in the 477
presence of specific respiratory chain inhibitors. 478
Inhibiting complex I by rotenone had no effect on the growth of A. fumigatus during 479
normoxia and resulted in only a very slight reduction of growth under hypoxic conditions in a 480
concentration-dependent manner (Fig. S7A). When complex I is inhibited, fungi and plants 481
are able to regenerate NAD+ via alternative NADH:ubiquinone oxidoreductases (53, 54). In 482
turn, inhibiting these alternative NADH dehydrogenases by flavone resulted in a strong 483
growth defect under both normoxic and hypoxic conditions (Fig. S7B). Even when combining 484
both inhibitors (rotenone and flavone) of the respiratory NAD+ regeneration system A. 485
fumigatus was still able to grow in normoxic and hypoxic atmospheres (Fig. S7C). Similarly, 486
as for complex I, inhibition of the complex II component succinate dehydrogenase by 487
disodium malonate, of complex V by oligomycin or of the transmembrane H+ carrier by 2,4-488
dinitrophenol resulted in a reduction in fungal growth, which was, however, largely 489
independent of the atmospheric O2-levels (Fig. S8, Fig. S9). 490
Hypoxia-specific effects were seen only for complexes III and IV. During normoxia the 491
lowest applied concentration of the complex III inhibitor antimycin A (5 µM) provoked only a 492
slight inhibitory effect on growth of A. fumigatus, but growth was severely impaired when 493
using the same concentration under hypoxic conditions (Fig. 6A). Similar results were 494
obtained when complex IV was inhibited by sodium azide (Fig. 6B). Under normoxia, growth 495
of A. fumigatus was reduced independent of the applied inhibitor concentration. In contrast, 496
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under hypoxic conditions sodium azide inhibited growth of A. fumigatus in a highly 497
concentration-dependent manner. At a concentration of 0.25 mM sodium azide, the wild type 498
did show a difference in sensitivity between normoxic and hypoxic conditions. This may be 499
explained by a compensatory increased activity of the alternativ oxidase AOX. 500
Many fungi are equipped with an AOX. It represents a terminal ubiquinol oxidase bypassing 501
complex III and IV of the electron transport chain (53, 54). The specific AOX-inhibitor 502
SHAM reduced growth of A. fumigatus wild type in a concentration-dependent manner under 503
both normoxic and hypoxic conditions (Fig. 7A). Remarkably, the mutant strain ∆srbA (Fig. 504
S10A), deleted in the hypoxia regulator SrbA, showed poor growth in presence of SHAM. To 505
investigate this further, Northern blot analysis was performed, which revealed an increased 506
expression of aox in the ∆srbA mutant under normoxic conditions (Fig. 7B). However, for 507
cytochrome C (cycA) no altered gene expression was observed between A. fumigatus wild 508
type and ∆srbA (Fig. 7B). This finding suggests that SrbA is directly or indirectly a negative 509
regulator of the aox gene under normoxic conditions. 510
Taken together, our data indicate that complex III and IV play an essential role in the 511
adaptation process towards hypoxic growth conditions. 512
Furthermore, our microarray data suggested that additional factors may contribute to the 513
assembly of respiratory complexes especially under hypoxic conditions. One candidate gene 514
encodes a protein which comprised a HIG-1 domain (PF04588, hypoxia inducible gene) and 515
revealed 32 % of identical amino acids to the Rcf2 protein from S. cerevisiae. In S. cerevisiae, 516
Rcf2 and its close homologue Rcf1, which is essential for hypoxic growth (55), are involved 517
in the assembly of respiratory supercomplexes (37). The hypoxic induction of the 518
corresponding homologue rcfB in A. fumigatus, prompted us to analyze whether its protein 519
product would also play a vital role in the assembly of these complexes during hypoxic 520
adaptation of A. fumigatus. The deletion of the rcfB gene (Fig. S10B) led to a general slight 521
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delay in growth, which was independent of the oxygen partial pressure. However, the 522
sporulation of the mutant strain rcfB was delayed after two days of incubation under mild 523
hypoxia (1 % pO2) compared to the wild-type strain (Fig. 8). 524
525
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Discussion 526
This study investigated the transcriptional response of A. fumigatus to hypoxia. Many 527
similarities in the adaptation to hypoxia can be found when comparing our results with 528
previous studies in Aspergillus species. As observed for A. nidulans and A. oryzae (18, 20) 529
intracellular trafficking processes and cytoskeletal transcripts were down-regulated under 530
hypoxic conditions, most probably in order to reduce ATP consumption. In contrast, genes 531
participating in the glyoxylate pathway were up-regulated under hypoxic conditions, whereas 532
the regulation of glycolytic genes was heterogenous (Dataset 1). In this aspect, the hypoxic 533
response of A. fumigatus resembles that of A. oryzae (18). The induction of the glyoxylate 534
cycle may help to avoid NADH accumulation by bypassing two NADH-producing steps of 535
the TCA cycle. The increased levels of transcripts contributing to amino acid transport are 536
consistent with results from A. nidulans (18) and may point to an increased production of 537
specific amino acids under hypoxia. 538
In a previous study, we characterized the adaptation of A. fumigatus to hypoxia in a batch-539
fermenter within a time window of 2 to 24 hours (defined here as short-term response). 540
Although the growth conditions were different (shorter incubation, batch fermentation, excess 541
of glucose) as described here, cell wall and sterol biosynthesis transcripts were up-regulated 542
(12). Similarly, with regard to respiration, complex IV associated transcripts were increased in 543
response to hypoxia. 544
Based on the obtained transcriptome data undertaken in this study, we tested our hypothesis 545
regarding the impact of alternative respiration in A. fumigatus during growth under long-term 546
hypoxic conditions. We aimed to elucidate the role of the single respiratory complexes in the 547
adaptation process of A. fumigatus to hypoxia by using specific respiratory inhibitors. In 548
addition, we analyzed the impact of alternative electron acceptors, such as nitrate and 549
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fumarate, on hypoxic growth of A. fumigatus in more detail by the generation of specific 550
knock-out strains. 551
Several microorganisms including different bacterial and fungal species make use of 552
alternative electron acceptors, such as nitrate, when oxygen is limited (41-45). Indeed, our 553
microarray experiment indicated that transcripts associated with nitrogen metabolism 554
significantly increased in response to hypoxic growth conditions. The transcript level of the 555
assimilatory nitrite reductase niiA was significantly induced, whereas the expression of the 556
nitrate reductase niaD was only slightly affected. Northern blot analysis revealed a slight 557
increase in transcription levels of niiA and niaD. However, our previous proteome study 558
analyzing the long-term response of A. fumigatus to hypoxia did not provide any evidence for 559
NAD+ regeneration via nitrate reduction (13). In line with this observation, the deletion of the 560
assimilatory nitrate and nitrite reductases did not affect growth under hypoxic conditions. This 561
suggests that in A. fumigatus, in contrast to A. nidulans, nitrate reduction does not 562
significantly support growth under hypoxia in A. fumigatus (22, 23). In F. oxysporum and 563
Cylindrocarpon tonkinense, membrane-bound, dissimilatory nitrate (dNaR) and nitrite 564
reductases (dNiR) were found to be part of a fungal denitrifying system, which is localized in 565
the mitochondria coupling with the electron transport chain to generate ATP under oxygen-566
limited conditions (42, 56, 57). Among them the fungal dNiR protein (nirK) of F. oxysporum, 567
which is a copper-containing nitrite reductase, was found to be the eukaryotic ortholog of the 568
bacterial counterpart. The C-terminal dNiR domain of nirK of F. oxysporum shares 76 % 569
identity with the respective amino acid sequence of the multicopper oxidase in A. fumigatus 570
(58). This protein was also found in our microarray analyses to be induced upon oxygen 571
limitation (Tab. 1). However, deletion mutants for this gene (AFUA_3G14950) were 572
indistinguishable from the wild type irrespective of the O2 levels (data not shown). 573
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The increased expression of transcripts associated with nitrate/nitrite reduction may also be 574
explained by a putative role of these enzymes in RNI detoxification. For example, a 575
contribution of nitrite reductase to NO management in an oxygen-limited environment has 576
been reported for bacteria like E. coli (59). Indeed, under low oxygen concentrations NO 577
levels may increase, since the mitochondrial respiratory chain complex is capable of 578
producing NO by reducing NO2
- (14, 15, 46-49). This oxygen-independent process, which is 579
activated under hypoxic or anoxic conditions, is catalyzed by the cytochrome c oxidase Cco 580
(14, 15). 581
Commonly, flavohemoglobins detoxify harmful NO within the fungal cell (60, 61). In our 582
microarray analyses the flavohemoglobin gene fhpA was found to be up-regulated by a factor 583
of 17 upon exposure to hypoxia, which is consistent with results obtained from previous 584
proteomic studies (12, 13). In the presence of nitrate, fhpA was induced over the entire time 585
course of hypoxia, whereas fhpA was only up-regulated within the first three hours of hypoxia 586
when using glutamine as nitrogen source. This observation was further confirmed by 587
fluorescence microscopy studies of a FhpA-eGFP fusion strain. Thus, we could demonstrate 588
that the expression of fhpA is induced independently of the available nitrogen source under 589
hypoxia. This favors the idea that elevated amounts of RNS are produced during hypoxia, 590
although their origin is currently unknown. 591
FhpA utilizes O2 and NAD(P)H to convert NO to NO3-. This oxygen dependence of NO 592
detoxification supports the idea that alternative, reductive enzymes, such as the nitrite 593
reductase NiiA, may take over the function of FhpA under hypoxia. Alternatively, the 594
induction of NiiA may be explained by a spontaneous oxidation of NO to NO2- (62). 595
However, there was only a moderate induction of niiA expression during hypoxia in the 596
Northern blot analysis. In addition, the niiA deletion mutant showed only sensitivity against 597
NO2- at acidic pH, but not in the presence of the NO-donor DETA-NO. 598
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None of the deletion mutants was growth-impaired in hypoxia. Thus, we conclude that the 599
increased expression of FhpA and nitrate and nitrite reductases was rather caused by NO-600
formation under hypoxia than by the hypoxic conditions itself. 601
Overall, in A. fumigatus, nitrate reduction does not seem to provide a mechanism to reload the 602
pool of NAD(P)+ or FAD
+ when O2 becomes limiting. Alternatively, the reduction of 603
fumarate to succinate is linked with the regeneration of NAD+ and may function as an 604
electron sink as described for Mycobacterium tuberculosis (63). Also, the fungal species A. 605
niger, A. oryzae and S. cerevisae produce elevated amounts of succinate under oxygen 606
limiting conditions (18, 52, 64). Gene expression of the cytosolic fumarate reductase frdA was 607
significantly induced in A. fumigatus during hypoxia, and succinate was detectable in the 608
supernatant of hypoxic batch cultures. 609
However, transcript levels of the mitochondrial fumarate reductase gene osmA remained 610
constant and thus were not affected by oxygen availability. These findings are consistent with 611
the reported regulation of the fumarate reductases frd1 and osm1 in S. cerevisae during 612
anaereobiosis (52). Contrary to S. cerevisae, in A. fumigatus deletion of fumarate reductases 613
as single or double knock-out strains had no effect on the growth phenotype under hypoxia 614
(51). Besides NAD+ regeneration, other functions have been suggested for fumarate 615
reductases, in particular redox balancing during anaerobiosis and the re-oxidation of flavin 616
cofactors (52, 65, 66). In line with this assumption, the A. fumigatus fumarate reductase 617
deletions strains ∆frdA, ∆osmA and ∆frdA/∆osmA were more susceptible to the reducing agent 618
TCEP, which affects the intracellular redox homeostasis. In summary, the A. fumigatus 619
fumarate reductase frdA may be highly up-regulated during hypoxia to maintain redox 620
homeostasis. Hence, our study supports the hypothesis that the expression of fumarate 621
reductase is up-regulated during hypoxia to reoxidize flavin cofactors and thus to keep 622
flavoenzymes in their oxidative state. 623
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Our data and previous studies on A. fumigatus and other fungi indicated an association of 624
functional mitochondria with the ability to adapt to low oxygen environments (11, 17, 67-70). 625
The up-regulation of several respiratory genes during long-term exposure to hypoxia is also 626
consistent with the increased production of components of the electron transport chain and 627
oxidative phosphorylation on the proteome level (12, 13). Besides the classical electron 628
transport chain via complex I to IV, fungi and plants posses also alternative routes: the 629
alternative NADH:ubiquinone oxidoreductase (a bypass of complex I) and the alternative 630
oxidase AOX which bypasses complex III and IV (53, 54). For this study we aimed at 631
investigating the role of each respiratory complex in the adaptation process of A. fumigatus 632
towards hypoxia. 633
Complex I transfers electrons from NADH to ubiquinone and thus is essential for the 634
regeneration of NAD+. Its specific inhibition with rotenone resulted in only a slight reduction 635
of growth of A. fumigatus under hypoxia. Interestingly, it was shown that rotenone only 636
partially inhibits the oxidation of NADH in A. fumigatus (71). Hence, the low inhibitory effect 637
of rotenone might be due to a compensatory reaction of alternative NADH dehydrogenases. 638
These can be specifically inhibited by flavone (71). In the presence of flavone, growth of A. 639
fumigatus was reduced independently of the level of oxygen. Even the combination of flavone 640
and rotenone had no significant effect on the hypoxic growth of A. fumigatus, indicating that 641
complex I and the alternative NADH:ubiquinone oxidoreductase are not absolutely essential 642
during hypoxic growth. This suggests that bypass routes exist under oxygen-limiting 643
conditions and electrons may enter the quinone pool independently of complex I. 644
The succinate dehydrogenase (complex II) oxidizes succinate to fumarate and transfers the 645
electrons to ubiquinone. Disodium malonate is a competitive inhibitor of complex II (54). 646
However, the inhibitory effect of disodium malonate on the growth of A. fumigatus was 647
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independent of the oxygen availability indicating a minor role of complex II in the adaptation 648
process of the fungi towards hypoxia. 649
Electrons of the ubiquinone pool can be either transferred to oxygen via cytochrome c by 650
complex III and IV or by the alternative oxidase (53, 54). We could demonstrate that the 651
alternative oxidase AOX is not essential for hypoxic growth since inhibition of AOX was 652
independent of the oxygen availability. In agreement with our data, hypoxic growth of the A. 653
fumigatus ∆aox strain was like the wild type (11). Interestingly, under normoxic conditions a 654
strain deleted for srbA was more susceptible to the alternative oxidase inhibitor SHAM. 655
Although transcript levels of aox were increased in the ∆srbA mutant, no altered gene 656
expression could be observed for cytochrome C. These data indicate that in ∆srbA the electron 657
transport proceeds via complex III and IV, but is also partially dependent on AOX illustrated 658
by the up-regulation of the corresponding aox gene in the mutant. Recently, it was 659
demonstrated that beside electron transport the alternative oxidase is also involved in the 660
oxidative stress response and macrophage killing of A. fumigatus (11). Remarkably, inhibition 661
of complex III by antimycin A provoked a strong growth defect of A. fumigatus during 662
hypoxia in a dose-dependent manner of the inhibitor, which was not observed under normoxic 663
conditions. Furthermore, in the presence of the complex IV inhibitor sodium azide growth of 664
A. fumigatus was significantly reduced under hypoxic compared to normoxic conditions. 665
Additionally, we could demonstrate that the transcripts of the cytochrome c oxidase subunit 666
coxVb and the rcf2-like gene rcfB increased during hypoxia. These findings are consistent 667
with the increased abundance of components of respiratory complex III and IV in our 668
proteome study of the long-term response of A. fumigatus to hypoxia (13). In this context it is 669
interesting to note that in S. cerevisiae coxVb is the more active isoform during hypoxia 670
whereas coxVa represents the dominating aerobic isoform (72). The proteins Rcf1 and Rcf2 671
are members of the hypoxia-induced gene 1 (Hig1) protein family and are involved in the 672
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assembly of the cytochrome c oxidase supercomplex in yeast (37). Although the A. fumigatus 673
∆rcfB deletion strain was still able to grow, reduced formation of conidia was observed during 674
mild hypoxia (1 % O2). Remarkably Rcf1 is required for hypoxic growth in yeast (55). 675
However, transcripts of the corresponding rcf1 homologue in A. fumigatus were not found to 676
be differentially regulated in our microarray data. 677
Overall, in A. fumigatus a functional electron transport chain obviously plays an essential role 678
for hypoxic growth. This particularly applies to respiratory complex III and IV, which might 679
be involved in sensing and adapting to hypoxia by post-translational protein modifications, 680
e.g. nitrotyrosination (16, 17). 681
In conclusion, this study analyzed the impact of aerobic respiration and reductive pathways on 682
hypoxic growth of A. fumigatus. Characterization of the A. fumigatus deletion strains ∆niiA, 683
∆niaD, ∆frdA, ∆osmA and ∆frdA/∆osmA revealed that neither nitrate nor fumarate were used 684
as alternative electron acceptors under oxygen limiting conditions. In accordance with this 685
finding we were able to demonstrate that a functional electron transport chain, especially 686
complex III and IV, is essential for the adaptation of A. fumigatus towards hypoxic growth 687
conditions. 688
689
Acknowledgements 690
This work was supported by funding from the German-Israeli Foundation for Scientific 691
Research and Development (GIF No. 996-47. 12/2008), the ERA-Net Pathogenomics project 692
OXYstress, and the International Leibniz Research School (ILRS) for Microbial and 693
Biomolecular Interactions as part of the DFG-funded excellence graduate school Jena School 694
for Microbial Communication (JSMC). 695
696
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Figures: 697
Fig. 1: Gene set enrichment (GSE) analysis of up- (A) and down-regulated (B) genes during 698
long-term exposure of A. fumigatus to hypoxia based on FunCat classification. 699
Fig. 2: Confirmation of the expresson of hypoxia-induced transcripts during cultivation 700
of A. fumigatus in an oxygen-controlled chemostat by Northern blot. A. fumigatus was 701
cultivated in a continuous culture under either normoxic or hypoxic conditions for 6 days. 702
Left panel: Northern hybridizations of up-regulated genes. RNA was isolated from normoxic 703
and hypoxic samples. 10 µg RNA were loaded per lane. rRNA bands are displayed as a 704
loading control. Right panel: Microarray fold-changes and p-value of the corresponding 705
genes. 706
Fig. 3: Regulation of flavohemoprotein fhpA during hypoxic growth conditions in 707
dependence of the nitrogen source. A) Northern blot analysis of fhpA: A. fumigatus 708
ATCC 46645 was cultivated under hypoxia in batch fermentation using nitrate or glutamine 709
as nitrogen source. RNA was isolated from samples taken after 0, 3, 6, 12 and 24 h of hypoxia 710
and 10 µg were loaded per lane. rRNA bands are displayed as a loading control. B) 711
Fluorescence studies of the FhpA-eGFP fusion strain: A. fumigatus FhpA-eGFP was 712
cultivated in AMM containing nitrate or glutamine as nitrogen source. After 14 h 713
precultivation under normoxic conditions cultures were incubated for a further 24 h at 1 % O2. 714
Using a constant exposure time fluorescence signals were monitored after 0, 3, 6, 12 and 24 h 715
of hypoxia. Size bar represents 20 µm. 716
Fig. 4: Growth of nitrate and nitrite reductase deletion strains under nitrosative stress 717
conditions. 5 µL aliquots of each strain were spotted in a serial 10-fold dilution on AMM 718
agar plates with 20 mM glutamine as primary nitrogen source and 0, 40 or 80 mM NaNO2 at 719
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pH 4.5 to impose nitrosative stress. Growth was documented after 48 h incubation at 37 °C 720
under normoxic conditions. 721
Fig. 5: A+B) Analysis of fumarate respiration during batch cultivation of A. fumigatus 722
under hypoxic conditions. A. fumigatus CEA17ΔakuBKU80
was cultivated under hypoxia in 723
batch fermentation. Samples were taken after 0, 3, 6, 12 and 24 h of hypoxia. A) Northern 724
blot analysis of frdA and osmA: 10 µg RNA were loaded per lane. rRNA was used as 725
loading control. B) Quantification of succinic acid in the supernatant. C + D) Influence of 726
TCEP on growth of fumarate reductase deletion strains. C) 5 µL aliquots of each strain 727
were spotted in a serial 10-fold dilution on AMM agar plates containing 0 mM or 10 mM 728
TCEP. D) 2x103 condia per well were cultivated in presence of 0 – 20 mM TCEP. Growth 729
differences were detected after 72 h incubation at 37 °C under normoxic conditions. 730
Fig. 6: Impact of the inhibition of complex III and IV on growth of A. fumigatus during 731
hypoxia. 5 µL aliquots of wild type and ΔsrbA strain were spotted in a serial 10-fold dilution 732
on AMM agar plates in the presence of A) antimycin A (0-20 µM) and B) sodium azide (0-1 733
mM). Growth differences were detected after 3 or 6 days incubation at 37 °C under normoxic 734
and hypoxic conditions. 735
Fig. 7: A) Impact of the inhibition of the alternative oxidase on growth of A. fumigatus 736
under hypoxic conditions. 5 µL aliquots of wild type and ΔsrbA strain were spotted in a 737
serial 10-fold dilution on AMM agar plates in the presence of SHAM (0-10 mM). Growth 738
differences were detected after 3 days incubation at 37 °C under normoxic and hypoxic 739
conditions. B) Northern blot analysis of A. fumigatus ΔsrbA: 100 mL AMM were 740
inoculated with 108 conidia of A. fumigatus wild type or ΔsrbA and incubated for 24 h at 37 741
°C and 200 rpm. RNA was isolated and 10 µg RNA were loaded per lane. rRNA bands are 742
displayed as a loading control. 743
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Fig. 8: Growth of A. fumigatus ΔrcfB under normoxic and hypoxic conditions. 744
Wild type and the deletion strains ΔrcfB and ΔsrbA were grown on AMM agar plates under 745
normoxia or hypoxia with a partial pressure of either 1% pO2 or 0.2 % pO2. Spores were 746
serially diluted in H2O and 5 µL containing 105 to 10
2 spores were inoculated on the plates. 747
Agar plates were incubated for 48 h at 37 °C and 5% CO2. 748
749
750
Table 1. Overview of the up-regulated transcripts associated with nitrate metabolism. 751
752
753
754
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Opin Microbiol 8:385-392. 756
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1:S77-81. 758
3. Latge JP. 2001. The pathobiology of Aspergillus fumigatus. Trends Microbiol 9:382-759
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4. Brakhage AA, Bruns S, Thywissen A, Zipfel PF, and Behnsen J. 2010. Interaction 761
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5. Brakhage AA. 2005. Systemic fungal infections caused by Aspergillus species: 763
epidemiology, infection process and virulence determinants. Cur Drug Targets 6:875-886. 764
6. Jain M, and Sznajder JI. 2005. Effects of hypoxia on the alveolar epithelium. Proc 765
Am Thorac Soc 2:202-205. 766
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responses to hypoxia: relevance to disease mechanisms. J Leukoc Biol 66:889-900. 768
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974
975
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degradation / modification of foreign
(exogenous) polysaccharides 3.81%
nuclear transport 8.30%
RNA transport 6.92%
energy conversion and
regeneration 4.15%
growth regulators /
regulation of cell size 4.15%
extracellular polysaccharide
degradation 4.15%
virulence, disease factors 9.34%
DNA topology 7.27%
enzyme activator 5.19%
amino acid/amino acid
derivatives transport 7.96%
polysaccharide
metabolism 16.61%
other 22.15%
modification by phosphorylation, dephosphorylation,
autophosphorylation 8.88%
guidance of longitudinal cell extension
(e.g. pollen tube guidance, axonal pathfinding) 2.32%
cell growth / morphogenesis 9.27%
cytoskeleton/structural
proteins 5.60%
anti-apoptosis 1.93%
ER to Golgi transport 3.47%
budding, cell polarity and
filament formation 9.46%
regulation of phosphate
metabolism 1.93%
osmotic and salt stress
response 3.86%
cell migration 2.70%
biosynthesis of phenylalanine 2.32%
osmosensing and response 3.28%
G1/S transition of mitotic cell cycle 2.51%
regulation of amino acid metabolism 2.12%
detoxification by modification 2.32%
actin cytoskeleton 4.25%
phospholipid metabolism 4.25%
spindle pole body/centrosome
and microtubule cycle 2.32%
cytokinesis (cell division) /
septum formation
and hydrolysis 5.79%
G-protein mediated
signal transduction 2.12%
purin nucleotide/nucleoside/
nucleobase metabolism 4.25%
GTP binding 4.25%
protein folding and stabilization 4.63%
other 6.18%
A
B
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21%
O2
0.2%
O2
fhpA
rRNA
frdA
osmA
coxVb
Fold
changep-value
17.09
321.22
6.19
3.68
3.79
1.51E-04
2.93E-04
1.20E-02
6.95E-03
5.85E-02rcfB
17.09 1.51E-04
6.19 1.20E-02
3.79 5.85E-02
37.73
4.69
9.94E-03
3.59E-044.69 3.59E-04
niiA
niaD
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A
fhpA
rRNA
0h 3h 6h 12h 24h
Time of hypoxia
N-source: Nitrate N-source: Glutamine
0h 3h 6h 12h 24h
Time of hypoxia
0 h
B
Nitrate
Glutamine
3 h 6 h 12 h 24 h
Time of hypoxia
20 µm
20 µm
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0 mM NaNO2
105 104 103 102
WT
∆niaD
∆niiA
40 mM NaNO2
105 104 103 102
80 mM NaNO2
105 104 103 102
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frdA
0h 3h 6h 12h 24h
rRNA
osmA
Batch cultivation
Time of hypoxiaA B
C
wt
∆frdA
∆osmA
∆frdA/
∆osmA
0 mM TCEP
105 104 103 102
10 mM TCEP
105 104 103 102
wt
∆frdA
∆osmA
∆frdA/
∆osmA
AMM
TCEP [mM]
0 2.5 5 10 15 20
D
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Complex III inhibitor - Antimycin A
WT
∆srbA
21 % O2 0.2 % O2
WT
∆srbA
WT
∆srbA
WT
∆srbA
0 µM Antimycin A
5 µM Antimycin A
10 µM Antimycin A
20 µM Antimycin A
105 104 103 102 105 104 103 102
3 days, 37°C
Complex IV inhibitor - Sodium azide (NaN3)
0 mM NaN3
0.25 mM NaN3
0.5 mM NaN3
1 mM NaN3
2•106
21 % O2
2•105 2•104 2•103 2•106
0.2 % O2
2•105 2•104 2•103
6 days 37°C
WT
∆srbA
WT
∆srbA
WT
∆srbA
WT
∆srbA
A B
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WT
∆srbA
WT
∆srbA
WT
∆srbA
WT
∆srbA
0 mM SHAM
2.5 mM SHAM
5 mM SHAM
10 mM SHAM
Alternative oxidase inhibitor - SHAM
21 % O2 0.2 % O2
105 104 103 102 105 104 103 102
A
WT ∆srbA
srbA
aox
cycA
rRNA
B
on March 27, 2020 by guest
http://ec.asm.org/
Dow
nloaded from
∆rcfB
WT
∆srbA
103 102104105
21 % O2
1 % O2
103 102104105
0.2 % O2
103 102104105
on March 27, 2020 by guest
http://ec.asm.org/
Dow
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Table 1. Overview of the up-regulated transcripts dealing with nitrate metabolism.
description accession ratio p-Value
Nitrate reductase NiaD AFUA_1G12830 +4.69 0.000359
Nitrite reductase NiiA AFUA_1G12840 +37.73 0.00994
Nitrate transporter CrnA AFUA_1G12850 +6.2 0.013
High affinity nitrate transporter NrtB AFUA_1G17470 +6.28 0.0923
Periplasmic nitrate reductase AFUA_3G15190 +4.29 0.00952
Nitrate Reductase AFUA_5G10420 +2.83 0.00242
Multicopper oxidase AFUA_3G14950 +19.46 0.0303
on March 27, 2020 by guest
http://ec.asm.org/
Dow
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