identification of hypoxia -inducible target genes of aspergillus … · 1 identification of hypoxia...

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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32

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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3. Latge JP. 2001. The pathobiology of Aspergillus fumigatus. Trends Microbiol 9:382-759

389. 760

4. Brakhage AA, Bruns S, Thywissen A, Zipfel PF, and Behnsen J. 2010. Interaction 761

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epidemiology, infection process and virulence determinants. Cur Drug Targets 6:875-886. 764

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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


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∆rcfB

WT

∆srbA

103 102104105

21 % O2

1 % O2

103 102104105

0.2 % O2

103 102104105


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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


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identification of hypoxia -inducible target genes of aspergillus … · 1 identification of hypoxia...

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