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

Abstract

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 (spz6) pathway. These data indicate that the innate immune system of Drosophila mediates the toxicity of fungal volatiles, especially via the Toll pathway.

Loss of the mammalian G-protein coupled receptor, G2A, modulates severity of invasive pulmonary aspergillosis

“…1 nmol/g lung, Table 1 ). To confirm that these oxylipins were truly derived from A. fumigatus , we then compared oxylipin production in mice infected with either a mutant deleted for all ppo enzymes (Δ ppoABC , TMN32.1) ( 31 ), the parental of TMN32.1 (CEA10ΔKU80, containing all 3 ppo genes) along with naïve and A1163 controls. 5,8-diHODE and 8-HODE were only detected in the lungs of the mice infected with wild type A. fumigatus ( Supplemental Figure 2 ).…”

“…A modified approach was used to confirm that the fungal-derived oxylipins were produced by A. fumigatus by comparing D2 p.i. mice infected with a CEA17Δ ku80 to a Δ ppoABC strain (TMN32.1) devoid of oxylipin production ( 31 ), along with naïve and A1163 controls (supplemental materials and methods).…”

“…A modified approach was used to confirm that the fungal-derived oxylipins were produced by A. fumigatus by comparing D2 p.i. mice infected with a CEA17Dku80 to a DppoABC strain (TMN32.1) devoid of oxylipin production (31), along with naïve and A1163 controls (supplemental materials and methods). The UPLC-MS/MS measurements were made using a Sciex 6500+ QTRAP system (Sciex, Redwood, CA, USA) hyphenated to a Waters Acquity UPLC system.…”

The utility of Drosophila melanogaster as a fungal infection model

“…Quantitative measurements of VOCs production showed that greater volumes of VOCs were secreted when A. fumigatus was cultivated at 37°C, than at the fly’s preferred incubation temperature of 25°C ( 57 , 89 ). In addition, exposure to VOCs resulted in varying levels of toxicity, ranging from mild to severe, including reduced speed and success rate of metamorphosis or death of 3rd instar larvae ( 57 , 90 ). Gas-chromatography mass spectrometry analysis of A. fumigatus VOCs detected isopentyl alcohol 1-octen-3-ol at the highest volume ( 57 ).…”