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. 2014 Sep;13(9):1241-53.
doi: 10.1128/EC.00084-14. Epub 2014 Aug 1.

Identification of Hypoxia-Inducible Target Genes of Aspergillus Fumigatus by Transcriptome Analysis Reveals Cellular Respiration as an Important Contributor to Hypoxic Survival

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Identification of Hypoxia-Inducible Target Genes of Aspergillus Fumigatus by Transcriptome Analysis Reveals Cellular Respiration as an Important Contributor to Hypoxic Survival

Kristin Kroll et al. Eukaryot Cell. .
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Abstract

Aspergillus fumigatus is an opportunistic, airborne pathogen that causes 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 a significant increase 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 and osmA), nitrogen metabolism (niaD and niiA), and respiration (rcfB). We show that the nitric oxygen (NO)-detoxifying flavohemoprotein gene 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 reductase 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 functional mitochondrial respiratory chain complexes are essential under these conditions. Inhibition studies indicated that primarily complexes III and IV play a crucial role in the hypoxic growth of A. fumigatus.

Figures

FIG 1
FIG 1
Gene set enrichment (GSE) analysis of upregulated (A) and downregulated (B) genes during long-term exposure of A. fumigatus to hypoxia based on FunCat classification.
FIG 2
FIG 2
Confirmation of the expression of hypoxia-induced transcripts during cultivation of A. fumigatus in an oxygen-controlled chemostat by Northern blotting. A. fumigatus was cultivated in a continuous culture under either normoxic or hypoxic conditions for 6 days. (Left panel) Northern hybridizations of upregulated genes. RNA was isolated from normoxic and hypoxic samples. Ten micrograms of RNA was loaded per lane. rRNA bands are displayed as a loading control. (Right panel) Microarray fold changes and P value of the corresponding genes.
FIG 3
FIG 3
Regulation of flavohemoprotein fhpA during hypoxic growth conditions in dependence of the nitrogen source. (A) Northern blot analysis of fhpA. A. fumigatus ATCC 46645 was cultivated under hypoxia in batch fermentation using nitrate or glutamine as the nitrogen source. RNA was isolated from samples taken after 0, 3, 6, 12, and 24 h of hypoxia, and 10 μg was loaded per lane. rRNA bands are displayed as a loading control. (B) Fluorescence studies of the FhpA-eGFP fusion strain. The A. fumigatus FhpA-eGFP strain was cultivated in AMM containing nitrate or glutamine as the nitrogen source. After 14 h of precultivation under normoxic conditions, cultures were incubated for a further 24 h at 1% O2. Using a constant exposure time, fluorescence signals were monitored after 0, 3, 6, 12, and 24 h of hypoxia. The size bar represents 20 μm.
FIG 4
FIG 4
Growth of nitrate and nitrite reductase deletion strains under nitrosative stress conditions. Five-microliter aliquots of each strain were spotted in a serial 10-fold dilution on AMM agar plates with 20 mM glutamine as the primary nitrogen source and 0, 40, or 80 mM NaNO2 at pH 4.5 to impose nitrosative stress. Growth was documented after 48 h of incubation at 37°C under normoxic conditions.
FIG 5
FIG 5
(A and B) Analysis of fumarate respiration during batch cultivation of A. fumigatus under hypoxic conditions. A. fumigatus CEA17ΔakuBKU80 was cultivated under hypoxia in batch fermentation. Samples were taken after 0, 3, 6, 12, and 24 h of hypoxia. (A) Northern blot analysis of frdA and osmA. Ten micrograms of RNA was loaded per lane. rRNA was used as the loading control. (B) Quantification of succinic acid in the supernatant. (C and D) Influence of TCEP on growth of fumarate reductase deletion strains. (C) Five-microliter aliquots of each strain were spotted in a serial 10-fold dilution on AMM agar plates containing 0 or 10 mM TCEP. (D) A total of 2 × 103 condia per well were cultivated in the presence of 0 to 20 mM TCEP. Growth differences were detected after 72 h of incubation at 37°C under normoxic conditions.
FIG 6
FIG 6
Impact of the inhibition of complexes III and IV on growth of A. fumigatus during hypoxia. Five-microliter aliquots of the wild-type and ΔsrbA strains were spotted in a serial 10-fold dilution on AMM agar plates in the presence of 0 to 20 μM antimycin A (A) and 0 to 1 mM sodium azide (B). Growth differences were detected after 3 or 6 days of incubation at 37°C under normoxic and hypoxic conditions.
FIG 7
FIG 7
(A) Impact of the inhibition of the alternative oxidase on growth of A. fumigatus under hypoxic conditions. Five-microliter aliquots of the wild-type and ΔsrbA strains were spotted in a serial 10-fold dilutions on AMM agar plates in the presence of SHAM (0 to 10 mM). Growth differences were detected after 3 days of incubation at 37°C under normoxic and hypoxic conditions. (B) Northern blot analysis of the A. fumigatus ΔsrbA strain. A 100-ml volume of AMM was inoculated with 108 conidia of the A. fumigatus wild-type or ΔsrbA strain and incubated for 24 h at 37°C and 200 rpm. RNA was isolated, and 10 μg RNA was loaded per lane. rRNA bands are displayed as a loading control.
FIG 8
FIG 8
Growth of the A. fumigatus ΔrcfB strain under normoxic and hypoxic conditions. The wild-type and ΔrcfB and ΔsrbA deletion strains were grown on AMM agar plates under normoxia or hypoxia with a partial pressure of either 1% pO2 or 0.2% pO2. Spores were serially diluted in H2O, and 5 μl containing 105 to 102 spores was inoculated on the plates. Agar plates were incubated for 48 h at 37°C and 5% CO2.

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