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Aspergillus fumigatus: Is it Radical? Bryan Prahl, Nancy Burritt, and James Burritt University of Wisconsin-Stout Menomonie, Wisconsin •Aspergillus fumigatus causes fatal infections in humans with weakened immune defense. •Literature indicates oxygen radicals guard against infection by A. fumigatus. •Our data suggest that under low-dose exposure, normal resistance to this infection relies on starvation of the pathogen. •We are collecting molecular evidence that starvation of A. fumigatus blocks infection by this organism . Methods: Fig. 2: Expression of αketoglutarate dehydrogenase (AKG) and isocitrate lyase (ICL) mRNA in conidia was compared using GAPDH as a normalizing gene. 1e7 conidia in a mouse lung lavage was directly compared to 1e7 conidia in medium containing glucose. Fig. 3: Removal of carbohydrate from medium results in blocked conidial development. Two hours following incubation at 37oC, a subset of conidia was washed and resuspended in medium lacking carbohydrate. Metabolic Pathways Transcriptional Analysis Starvation of fungus blocks development 3.9 Figure 2 key points: Figure 1 key points: • Kreb’s cycle vs. glyoxylate cycle • Kreb’s is preferred due to more energy being produced. • We are examining two enzymes near the split of the pathways. • Gene expression analysis is by reverse transcriptase PCR • Isocitrate lyase was upregulated in conidia bound in immune cells collected from the lung of a mouse. • α-ketogluterate is upregulated in free conidia collected from medium containing glucose. • Enzymes are inversely related. conidial diameters (microns) Overview: removal of carbohydrate 3.7 continuous CHO removed 3.5 3.3 3.1 2.9 2.7 2.5 0 1 2 3 hours incubation Figure 3 key points: 4 Summary •Mechanism of resistance under lowdose exposure is not known. •Removal of carbohydrates stops conidial swelling in response to starvation. •ICL and AKG are inversely related in respect to the metabolic pathway. • Removing carbohydrates stops conidial swelling. • Same behavior is observed in macrophages. • Starvation response from •Future analysis will involve studying conidia includes halted reduced glucose swelling. concentrations to reduce germination and infection in humans.