Volatile Organic Compounds Produced by Human Pathogenic Fungi Are Toxic to Drosophila melanogaster

Abstract

Volatile organic compounds (VOCs) are low molecular mass organic compounds that easily evaporate at room temperature. Fungi produce diverse mixtures of VOCs, some of which may contribute to "sick building syndrome," and which have been shown to be toxigenic in a variety of laboratory bioassays. We hypothesized that VOCs from medically important fungi might be similarly toxigenic and tested strains of Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, Cryptococcus gattii, and Saccharomyces cerevisiae in a Drosophila melanogaster eclosion bioassay. Fungi were grown in a shared microhabitat with third instar larvae of D. melanogaster such that there was no physical contact between flies and fungi. As the flies went through metamorphosis, the numbers of larvae, pupae, and adults were counted daily for 15 days. After 8 days, ~80% of controls had eclosed into adults and after 15 days the controls yielded 96-97% eclosion. In contrast, eclosion rates at 8 days were below 70% for flies exposed to VOCs from six different A. fumigatus strains; the eclosion rate at 15 days was only 58% for flies exposed to VOCs from A. fumigatus strain SRRC 1607. When flies were grown in a shared atmosphere with VOCs from S. cerevisiae, after 15 days, 82% of flies had eclosed into adults. Exposure to the VOCs from the medically important yeasts Candida albicans, Cryptococcus neoformans, and Cryptococcus gattii caused significant delays in metamorphosis with eclosion rates of 58% for Candida albicans, 44% for Cryptococcus neoformans, and 56% for Cryptococcus gattii. Using gas chromatography-mass spectrometry, the VOCs from the most toxic and least toxic strains of A. fumigatus were assayed. The two most common VOCs produced by both strains were 1-octen-3-ol and isopentyl alcohol; however, these compounds were produced in 10-fold higher concentrations by the more toxic strain. Our research demonstrates that gas phase compounds emitted by fungal pathogens may have been overlooked as contributing to the pathogenicity of medically important fungi and therefore deserve more scrutiny by the medical mycology research community.

Keywords: Aspergillus fumigatus; Candida albicans; Cryptococcus gattii; Cryptococcus neoformans; Drosophila melanogaster; Virulence; volatile organic compounds.

Figure 1

The bottle-plate microhabitat for placing Drosophila developmental stages and fungal cultures in a shared atmosphere without physical contact between the flies and fungi.

Figure 2

Percent living larval, pupal, and adult stages of 15 days of continuous exposure to VOCs emitted by six strains of A. fumigatus. The numbers of VOC-exposed larvae were compared to controls and the statistical differences were calculated on the 4th day: (A) Fungi were pre-grown at 25°C, (B) Fungi were pre-grown at 37°C. Where *represents the significant difference between controls and A. fumigatus exposed flies (*P < 0.05, **P < 0.005, ***P < 0.0005 and NS = Non-significant). A dashed vertical line denotes the day for statistical analysis. The numbers of VOC-exposed pupae were compared to controls and the statistical differences were calculated on the 8th day: (C) Fungi were pre-grown at 25°C, (D) Fungi were pre-grown at 37°C. The numbers of VOC-exposed adults were compared to controls and the statistical differences were calculated on the 15th day: (E) Fungi were pre-grown at 25°C; (F) Fungi were pre-grown at 37°C. Comparative analysis of percent of eclosion adult flies treated by VOCs emitted from six different strains of A. fumigatus (N = 90) at 7th, 10th, and 15th days at either 25°C (G) or 37°C (H).

Figure 3

Percent larval, pupal, and adults of 15 days of continuous exposure to VOCs emitted by three yeast species and one S. cerevisiae strain. The numbers of VOC-exposed larvae were compared to controls and the statistical differences were calculated on the 4th day: (A) different strains were pre-grown at 25°C, (B) different strains were pre-grown at 37°C. Where *represents the significant difference between controls and VOC-exposed flies (*P < 0.05, **P < 0.005, ***P < 0.0005 and NS = Non-significant). A dashed vertical line denotes the day for statistical analysis. The numbers of VOC-exposed pupae were compared to controls and the statistical differences were calculated on the 8th day: (C) different strains were pre-grown at 25°C, (D) different strains were pre-grown at 37°C. The numbers of VOC-exposed adults were compared to controls and the statistical differences were calculated on the 15th day: (E) different strains were pre-grown at 25°C, (F) different strains were pre-grown at 37°C. Comparative analysis of percent of eclosion adult flies treated by VOCs emitted by three yeast species and one S. cerevisiae strain (N = 90) grown at 7th, 10th, and 15th days at either 25°C (G) or 37°C (H).

Figure 4

GC-MS analyses of VOCs from A. fumigatus strains SRRC 1607 and 1592. (A) VOCs emitted by SRRC 1607 cultivated for 5 day at 25°C. (B) VOCs emitted by SRRC 1607 cultivated for 3 day at 37°C. (C) VOCs emitted by SRRC 1592 cultivated for 5 day at 25°C. (D) VOCs emitted by SRRC 1592 cultivated for 3 days at 37°C.