Sniffing Out the Hypoxia Volatile Metabolic Signature of Aspergillus fumigatus

Background: Aspergillus fumigatus is responsible for the life-threatening infection invasive aspergillosis (IA) that is associated with high mortality in immunocompromised individuals. Current diagnostics for IA are either highly invasive or suffer from inadequate sensitivity and/or specificity. Improvement of IA diagnostic assays requires a better understanding of fungal metabolism inside the host. The infection microenvironment becomes progressively depleted of oxygen during IA. Thus, metabolites associated with hypoxia metabolism may yield to novel diagnostic biomarkers. Here, we report on the volatile metabolites emitted from A. fumigatus batch cultures under normoxia and hypoxia conditions.

Methods: A. fumigatus strain CEA10 was incubated in glucose minimal medium at 37 °C under either normoxia (21% O2, 0.04% CO2) or hypoxia (0.2% O2, 5% CO2) growth conditions. Volatile molecules from the supernatants of early (1 h) and late (8 h) cultures were collected using headspace solid-phase microextraction (HS-SPME) and analyzed using two-dimensional gas chromatography-time-of-flight mass spectrometry (GCxGC-TOFMS).

Results: We identified 10 volatile compounds that are significantly more abundant in late hypoxia cultures compared with all other experimental conditions. Among these compounds, we identified ethanol and acetaldehyde, which are known to result from hypoxia metabolism of glucose by A. fumigatus. In addition, we detected 2-propanol, which can be generated by the reduction of acetone via alcohol dehydrogenase, as well as 2,3-butanedione (diacetyl) and 3-hydroxy-2-butanone (acetoin), which results from the catabolism of acetolactate. Transcriptomic data collected from normoxia and hypoxia cultures validates our volatilomic findings through the identification of putative catabolic pathways that are upregulated during hypoxia growth. Diacetyl is known to be produced by bacteria as a pH-neutral fermentative byproduct to prevent lethal acidification, and we speculate that A. fumigatus may use the same pathway.

Conclusion: The composition of A. fumigatus’ volatile metabolome changes according to the oxygen tension in the environment. We measured hypoxia-associated compounds, ethanol and acetaldehyde which have been previously identified as byproducts of hypoxia metabolism in this organism. 2-propanol, 2, 3-butanedione and 3-hydroxy-2-butanone represent novel metabolites that suggests the existence of other catabolic pathways activated in A. fumigatus under hypoxia growth conditions.