Comment[ArrayExpressAccession]	E-MTAB-2699
MAGE-TAB Version	1.1
Investigation Title	Long-term adaptation to hypoxic conditions - Febit transcriptome analysis
Comment[Submitted Name]	Long-term adaptation to hypoxic conditions - Febit transcriptome analysis
Experiment Description	Aspergillus fumigatus is an opportunistic, airborne pathogen causing invasive aspergillosis in immunocompromised patients. During the infection process A. fumigatus is challenged by hypoxic microenvironments occurring in inflammatory, necrotic tissue. To gain further insights into the adaptation mechanism, A. fumigatus was cultivated in an oxygen-controlled chemostat under hypoxic and normoxic conditions. Transcriptome analysis revealed significant increases in transcripts associated with cell wall polysaccharide metabolism, amino acid and metal ion transport, nitrogen metabolism and glycolysis. A concomitant reduction in transcript levels was observed with cellular trafficking and G-protein coupled signaling. To learn more about the functional roles of hypoxia-induced transcripts we deleted A. fumigatus genes putatively involved in reactive nitrogen species detoxification (fhpA), NAD+ regeneration (frdA, osmA) nitrogen metabolism (niaD, niiA) and respiration (rcfB). We show that the NO-detoxifying flavohemoprotein fhpA is strongly induced by hypoxia independent of the nitrogen source, but is dispensable for hypoxic survival. By deleting the nitrate reductase gene niaD, the nitrite reductase gene niiA and the two fumarate reductases genes frdA and osmA, we found that alternative electron acceptors such as nitrate and fumarate do not have a significant impact on growth of A. fumigatus during hypoxia, but that functional mitochondrial respiratory chain complexes are essential under these conditions. Inhibition studies indicated that primarily complex III and IV play a crucial role in the hypoxic growth of A. fumigatus.
Experimental Design	growth condition design	reference design	co-expression_design
Comment[AEExperimentType]	transcription profiling by array
Experimental Factor Name	growth condition
Experimental Factor Type	growth condition
Quality Control Type	biological replicate
Quality Control Term Source REF	EFO
Public Release Date	2014-07-01
Person Last Name	Schmidt-Heck	Kniemeyer
Person First Name	Wolfgang	Olaf
Person Mid Initials
Person Email	wolfgang.schmidt-heck@hki-jena.de	olaf.kniemeyer@hki-jena.de
Person Phone
Person Address
Person Affiliation
Person Roles	submitter	investigator
PubMed ID
Publication Author List	Kristin Kroll, Vera PM-BM-^Jhtz, Falk Hillmann, Yakir Vaknin, Wolfgang Schmidt-Heck, Martin Roth, Ilse D. Jacobsen, Nir Osherov and Olaf Kniemeyer
Publication Title	Identification of hypoxia-inducible targets of Aspergillus fumigatus by transcriptome analysis reveals respiratory genes as important contributors to hypoxic survival
Publication Status	under review
Protocol Name	P-MTAB-39791	P-MTAB-39792	P-MTAB-39793	P-MTAB-39794	P-MTAB-39795	P-MTAB-39796	P-MTAB-39797
Protocol Type	growth protocol	treatment protocol	nucleic acid extraction protocol	nucleic acid labeling protocol	nucleic acid hybridization to array protocol	array scanning protocol	normalization data transformation protocol
Protocol Description	A. fumigatus was grown in Aspergillus minimal medium (AMM)  containing 60 mM glucose and 70 mM NaNO3 as sole carbon and nitrogen source, respectively. To study the long-term response of A. fumigatus to hypoxic growth conditions, A. fumigatus strain ATCC 46645 was grown in a glucose-limited oxystat (chemostat with constant oxygen partial pressure) in a continuous culture. Batch fermentation was used to analyze the short-term response of A. fumigatus to hypoxic growth conditions.  For expression analysis of the flavohemoprotein fhpA either 70 mM NaNO3 or 20 mM L-glutamine were used as nitrogen sources in batch cultivations of A. fumigatus.	To study the effect of long-term exposure to hypoxia on the transcriptional profile, A. fumigatus  was  cultivated in a chemostat, which allowed to vary the oxygen partial pressure (21 % pO2 and 0.21 % pO2), while all other cultivation parameters such as glucose concentration, pH and temperature were kept constant. The dilution rate applied was 0.08 h-1. Steady-state growth was reached after between 3-4 days and the biomass was harvested after 5-6 days followed by RNA extraction. 	For microarray analyses, total RNA was isolated from chemostat cultures. Frozen mycelium of A. fumigatus ATCC 46645 was ground to a fine powder and 100 mg were used for total RNA isolation using the Qiagen M-RRNeasy Mini KitM-S according to the manufacturerM-Us instructions. Following DNase treatment (TurboDNA-free kit, Ambion, Germany) the quantity and quality of RNA preparations were determined spectrophotometrically with a nano-drop (Thermo Fisher Scientific, Germany).	Febit uses the MessageAmpTMII-Biotin Enhanced RNA Kit from Ambion for the labeling of RNA for mRNA expression analysis. The kit provides a transcription of RNA in cDNA, following a transcription in cRNA while enrichment of all nucleic acid molecules is included. For each array 1 M-5g of total RNA was labeled according to the manufacturerM-Us instructions.	 After, labeled samples were dried in a speed-vac and fragmented with a fragmentation buffer (see febit protocol 20).  Finally, febits proprietary standard Hybridization Buffer (20 M-5l per array) was added. For each array the RNA was suspended in febitM-Us proprietary mRNA Hybridization Buffer (25 M-5l per array). Hybridization was done automatically for 16h at 45?C using the Geniom RT-Analyzer.	For maximum sensitivity, febit used biotin and its detection with streptavidin-phycoerythrin(SAPE), in combination with febitM-Us consecutive Signal Enhancement (CSE) procedure. For a more detailed description please read febit protocol 010. The feature recognition (using Cy3 filter set) and signal calculation were done automat-ically within milliseconds.  The Geniom Technology showed accurate detection of mRNA profiles.	All data analyses of the FEBIT microarrays were performed using M-TLIMMAM-U (Linear Models for Microarray Data) packages  of the Bioconductor Software. Background correction was performed using the intensities of blank probes which consist of only one single M-STM-S nucleotide. The median background intensity is subtracted from spot intensity. After converting any negative values to low positive value, signal intensities were log2-transformed, and duplicate spots were averaged. The obtained data were processed using quantile normalization.
Protocol Term Source REF	EFO	EFO	EFO	EFO	EFO	EFO	EFO
Term Source Name	EFO	ArrayExpress
Term Source File	http://www.ebi.ac.uk/efo	http://www.ebi.ac.uk/arrayexpress
SDRF File	E-MTAB-2699.sdrf.txt