Transcript Prahl NCUR

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.