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. 2011 May 6;10(5):2508-24.
doi: 10.1021/pr1012812. Epub 2011 Mar 29.

Analysis of the Aspergillus Fumigatus Proteome Reveals Metabolic Changes and the Activation of the Pseurotin A Biosynthesis Gene Cluster in Response to Hypoxia

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Analysis of the Aspergillus Fumigatus Proteome Reveals Metabolic Changes and the Activation of the Pseurotin A Biosynthesis Gene Cluster in Response to Hypoxia

Martin Vödisch et al. J Proteome Res. .
Free PMC article

Abstract

The mold Aspergillus fumigatus is the most important airborne fungal pathogen. Adaptation to hypoxia represents an important virulence attribute for A. fumigatus. Therefore, we aimed at obtaining a comprehensive overview about this process on the proteome level. To ensure highly reproducible growth conditions, an oxygen-controlled, glucose-limited chemostat cultivation was established. Two-dimensional gel electrophoresis analysis of mycelial and mitochondrial proteins as well as two-dimensional Blue Native/SDS-gel separation of mitochondrial membrane proteins led to the identification of 117 proteins with an altered abundance under hypoxic in comparison to normoxic conditions. Hypoxia induced an increased activity of glycolysis, the TCA-cycle, respiration, and amino acid metabolism. Consistently, the cellular contents in heme, iron, copper, and zinc increased. Furthermore, hypoxia induced biosynthesis of the secondary metabolite pseurotin A as demonstrated at proteomic, transcriptional, and metabolite levels. The observed and so far not reported stimulation of the biosynthesis of a secondary metabolite by oxygen depletion may also affect the survival of A. fumigatus in hypoxic niches of the human host. Among the proteins so far not implicated in hypoxia adaptation, an NO-detoxifying flavohemoprotein was one of the most highly up-regulated proteins which indicates a link between hypoxia and the generation of nitrosative stress in A. fumigatus.

Figures

Figure 1
Figure 1
2D gel electrophoresis of protein extracts of A. fumigatus grown under hypoxic (Cy5, green) and normoxic conditions (Cy3, purple). Proteins were stained with the difference in gel electrophoresis (DIGE) labeling technique. The orientation of the IEF is indicated. The spot numbers refer to differentially synthesized proteins under hypoxic growth conditions (see Table 1).
Figure 2
Figure 2
2D gel electrophoresis of mitochondrial protein extracts of A. fumigatus grown under hypoxic (Cy5, green) and normoxic conditions (Cy3, purple). Proteins were stained with the difference in gel electrophoresis (DIGE) labeling technique. The orientation of the IEF is indicated. The spot numbers refer to differentially synthesized proteins under hypoxic growth conditions (see Table 2).
Figure 3
Figure 3
Analyses of the protein complexes of the mitochondrial respiration chain of A. fumigatus by two-dimensional Blue-native/SDS PAGE under hypoxic conditions. Proteins were visualized by Coomassie staining. Designation of resolved protein complexes are given on top of the gel. The spot numbers refer to proteins listed in Table 3.
Figure 4
Figure 4
Physiological adaptation of A. fumigatus to hypoxic growth condition with regard to modifications in the respiratory activity and the amount of mitochondria in the hyphae. (A) Oxygen consumption rate of A. fumigatus hyphae grown under normoxic (21% pO2) and hypoxic (0.2% pO2) conditions. The oxygen consumption rate of A. fumigatus grown in hypoxia is significantly increased compared to that of A. fumigatus cultivated under normoxic environments (p < 0.01). Error bars indicate standard deviations of the means. (B) Determination of the amount of A. fumigatus mitochondria by Mito Tracker Red fluorescence staining. CLSM fluorescence (FL), brightfield (BF) and overlay (OV) images showing a clearly elevated amount of mitochondria in the mycelium of hypoxic (0.2% pO2) cultivated A. fumigatus in comparison to A. fumigatus grown under normoxia (21% pO2). All scale bars represent 5 μm length.
Figure 5
Figure 5
O2-dependent changes in the mycelial color and in the intracellular concentration of heme and metals in A. fumigatus. (A) Color of the mycelium of A. fumigatus turned from whitish to reddish due to the reduction of the oxygen partial pressure from 21 to 0.2% in the medium. (B) Differences in the metal (left y-axis) and heme (right y-axis) content of A. fumigatus mycelia grown under hypoxic (dark gray bar) in comparison to normoxic (light gray bar) conditions. The metals iron, zinc and copper and the prosthetic group heme are significantly (p < 0.05 * or p < 0.01**) enriched in A. fumigatus mycelia under hypoxia. Error bars indicate standard deviations of the means.
Figure 6
Figure 6
Intracellular concentrations of the pyridine nucleotides of A. fumigatus under normoxia (light gray bar) and hypoxia (dark gray bar). Hypoxic growth conditions led to a significant (p < 0.05 * or p < 0.01**) increase of the NADP+ and NADPH concentration (left y-axis) and to a complete conversion of the NADP+/NADPH ratio (right y-axis) in A. fumigatus. Error bars indicate standard errors of the means.
Figure 7
Figure 7
Induction of the pseurotin A biosynthesis gene cluster as well as the biosynthesis of pseurotin A under hypoxic, glucose-limited growth conditions in A. fumigatus. (A) Northern blot analysis and organization of the pseurotin A biosynthesis gene cluster. Total RNA was isolated from A. fumigatus grown under hypoxic and normoxic conditions. rRNA bands are shown as control. All genes of this cluster are highly induced under hypoxia in comparison to normoxia. Arrows indicate open reading frames. H1+2: hydrolases; MT: methyl transferase; P450: cytochrome P450 oxidoreductase; GST: glutathione S-transferase. (B) HPLC profiles of supernatant of the cultures of A. fumigatus grown under hypoxic and normoxic conditions. Commercially available pseurotin A was used as standard. Hypoxic growth conditions induced the biosynthesis of pseurotin A in A. fumigatus. (C) Northern blot analysis of the expression of srbA in A. fumigatus grown under normoxic and hypoxic conditions. rRNA bands are shown as control.
Figure 8
Figure 8
Quantitative Real Time-PCR to determine the relative expression of the hybrid PKS/NRPS and methyl transferase genes of the pseurotin A biosynthesis gene cluster in comparison to the control gene act1 (AFUA_6G04740) under normoxic and hypoxic conditions. (A) Relative expression of the genes for the hybrid PKS/NRPS and methyl transferase of the pseurotin A biosynthesis gene cluster in vitro and in the lung of infected mice. For the in vitro tests A. fumigatus was grown under hypoxic and normoxic conditions and the total RNA was subsequently isolated from the mycelium. For the in vivo data, mouse infection experiments were performed and the total RNA was isolated from two infected lungs. (B) Northern blot analysis of the standard gene act1. Ten μg of total RNA of A. fumigatus mycelium grown during hypoxia (0.2% pO2) and normoxia (21% pO2) was analyzed.

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