Volatile organic compounds that are produced from human pathogenic fungi are toxic to the genetic model drosophila melanogaster
Creators: name: Almaliki, Hadeel
Publisher: name: No Publisher Supplied
Year: 2020

description: Volatile organic compounds (VOCs) are organic compounds with low molecular mass and the ability to vaporize easily at room temperatures. Fungi produce many VOCs that vary in type and amount depending on producing species, as well as factors that influence growth such as temperature, substrate, moisture, pH, and other parameters. In nature, fungal VOCs are used as ecological signaling agents. In the built environment, for individuals who live in water damaged and mold infested houses, they have been implicated as possible contributors to “sick building syndrome” Finally, fungal VOCs have found practical applications in medical mycology as indirect assays for determining the presence of fungal growth. In this thesis, I have postulated that the VOCs emitted by medically important fungi may contribute to the pathogenicity of human fungal pathogens. Using a Drosophila melanogaster bioassay, third instar larvae were placed in a shared atmosphere with growing cultures of Aspergillus fumigatus, Cryptococcus gatti, Cryptococcus neoformans, Candida albicans and Saccharomyces cerevisiae. In some cases, the human pathogenic fungi were pre-cultured at 25oC and in other cases they were pre-cultured at 37oC. Control larvae were incubated in the absence of fungal VOCs. The number of larvae, pupae and adults was counted over a 15-day period and the effect of fungal VOCs on stages of metamorphosis, time to eclosion, and fly viability was determined. In general, exposure of larvae to VOCs from human pathogenic fungi slowed the rate of metamorphosis, delayed eclosion, and caused toxicity. In some cases, morphological abnormalities were observed. These harmful effects were more pronounced when fungi were pre-grown at 37◦C than at 25◦C. Larvae grown in the presence of VOCs from Saccharomyces cerevisiae had metamorphotic and eclosion rates that were like controls. The VOCs from the environmental strain of A. fumigatus had the highest toxicity to the developmental stages of Drosophila when it was grown at either 25°C or 37°C. Exposure to VOCs from Cryptococcus neoformans caused more delays on fly metamorphosis and more toxicity than did exposure to VOCs from the six A. fumigatus strains tested. Using purge and trap gas chromatography-mass spectrometry (GC-MS), the VOCs from the most toxic and least toxic strains of Aspergillus fumigatus were assayed. The most toxic strains produced high levels of 1-octen-3-ol, an eight carbon alcohol that previously has been determined to be toxic in low concentrations to Drosophila flies, Arabidopsis plants, human embryonic cell cultures, and to cause nasal irritation in trials with human subjects. In conclusion, I postulate that VOCs from medically important fungal species may be acting as virulence factors during human infections, thereby enhancing the pathogenic effects of these species. The second section of the dissertation focused on Aspergillus fumigatus strains that did or did not carry defects in their oxylipin pathways. Lipoxygenase (LOX) genes and oxylipins are involved in biosynthesis of several metabolites that affect various reproductive functions in filamentous fungi that are mediated by VOCs. A. wild type A. fumigatus strain and a “near wild type strain” with normal oxylipin genes were compared with A. fumigatus mutants blocked in the lipoxygenase pathway. The Drosophila bioassay with third instar larvae was used to detect delays in metamorphosis, eclosion and possible toxicity to flies. Exposure of larvae to VOCs produced by A. fumigatus wild type and overexpressed LoxB strain caused more delays on fly metamorphosis than did exposure to VOCs from A. fumigatus carrying blocks in the lipoxygenase pathway. There were no significant effects shown in the presence of arachidonic acid on the metamorphosis of the fruit fly compared with cultures lacking arachidonic acid. GC-MS analysis showed the wild type strain produced more abundant VOCs in higher concentrations than did the triple LOX mutant which released fewer VOCs in lower concentrations. VOCs produced by the A. fumigatus wild type strain AF293 included 1-octen-3-ol, 1-butanal, 1-octen, decanoic acid, lauric acid, myristic acid, and palmitic acid. These VOCs were not detected from the LOX triple mutant strain. The final section of the dissertation studied the most toxigenic eight carbon compound 1-octen-3-ol. I hypothesize 1-octen-3-ol is of distinct importance as a toxigenicity factor. Toxigenicity is usually defined as the ability of a pathogenic organism to product injurious substances that damage the host. In order further to investigate this hypothesis, I tested low concentrations of chemical standards of three eight carbon volatiles (1-octen-3-ol, 3-octanone and 3-octanol) characteristic of fungal metabolism against adult male Drosophila flies in order to determine their impact on fly survival. Two strains of Drosophila, one with red eyes and one with white eyes, that were wild type for immune pathway genes served as controls. In addition, strains that carried blocked mutations in the nitric oxide pathway, the Toll pathway, the Imd pathways, and a double mutant strains with blocks in both the Imd and Toll pathway were tested. Volatile phase 1-octen-3-ol and 3-octanone at 0. 5µL/L were toxic to almost all the strains after 24 hours. Oregon R (red-eyed, wild type) strain was less susceptible than wild type, white eyed strain (W1118) to all three volatile compounds at both concentrations. All strains showed high susceptibility to the high level of 1-octen-3-ol in the span of only two hours. Of the three compounds tested, 3-octanol at 0. 1µL/L was the least toxic to mutant and wild type strains. White eyed flies carrying the NOS mutation were more resistant to volatiles than were the white eyed control strains. The Relish E20 white-eyed, mutant strain was more susceptible than the spz6 red-eyed mutant strain to volatile phase 1-octen-3-ol at 0.1µL/L. The double mutant strain (red-eyed Relish E20 spz6) showed greater resistance to the presence of the volatile phase compounds than did either single mutant. This latter finding implies that the toxicity of 1-octen-3-ol and other VOCs may be related to aspects of the innate immune system that cause negative side effects on fly physiology; when the immune system is impaired, these negative side effects do not occur and the flies have increased survival in the presence of VOCs. In summary, the Drosophila bioassay has been used to show that VOCs from several medically important fungi are toxigenic. Strains of A. fumigatus that were blocked in lipoxygenase activity emitted fewer VOCs and had less toxicity. Presence of the eight-carbon volatile, 1-octen-3-ol was correlated with higher levels of toxicity in the Drosophila bioassay. VOCs from growing fungi can be considered toxigenic factors that contribute to pathogenic profiles of medically important fungi. To our knowledge, this is the first report that correlates fungal VOCs such as 1-octen-3-ol to the virulence of human medical pathogens.