Mutational Analysis of Aspergillus fumigatus Volatile Oxylipins in a Drosophila Eclosion Assay.

Aspergillus fumigatus is a ubiquitous opportunistic pathogen. We have previously reported that volatile organic compounds (VOCs) produced by A. fumigatus cause delays in metamorphosis, morphological abnormalities, and death in a Drosophila melanogaster eclosion model. Here, we developed A . fumigatus deletion mutants with blocked oxylipin biosynthesis pathways (∆ ppoABC ) and then exposed the third instar larvae of D. melanogaster to a shared atmosphere with either A. fumigatus wild-type or oxylipin mutant cultures for 15 days. Fly larvae exposed to VOCs from wild-type A . fumigatus strains exhibited delays in metamorphosis and toxicity, while larvae exposed to VOCs from the ∆ ppoABC mutant displayed fewer morphogenic delays and higher eclosion rates than the controls. In general, when fungi were pre-grown at 37 °C, the effects of the VOCs they produced were more pronounced than when they were pre-grown at 25 °C. GC-MS analysis revealed that the wild-type A. fumigatus Af293 produced more abundant VOCs at higher concentrations than the oxylipin-deficient strain Af293∆ ppoABC did. The major VOCs detected from wild-type Af293 and its triple mutant included isopentyl alcohol, isobutyl alcohol, 2-methylbutanal, acetoin, and 1-octen-3-ol. Unexpectedly, compared to wild-type flies, the eclosion tests yielded far fewer differences in metamorphosis or viability when flies with immune-deficient genotypes were exposed to VOCs from either wild-type or ∆ ppoABC oxylipin mutants. In particular, the toxigenic effects of Aspergillus VOCs were not observed in mutant flies deficient in the Toll ( spz 6 ) pathway. These data indicate that the innate immune system of Drosophila mediates the toxicity of fungal volatiles, especially via the Toll pathway.