Transcript Aspergillus fumigatus: Growth and Virulence

Aspergillus fumigatus:
Growth and Virulence
Judith C. Rhodes, Ph.D.
University of Cincinnati
Cincinnati, OH, USA
[email protected]
Aspergillus fumigatus
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Ubiquitous organism.
Most commonly reported
opportunistic
hyphomycete.
Important in compost
cycle.
How did this grass eater
become an opportunistic
pathogen?
A. fumigatus: Compost to Man
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Life is very competitive in a compost pile.
What are some of the growth characteristics that enable A.
fumigatus to be successful in the environment that may also
allow it to be an opportunistic pathogen?
Growth Traits and Increased Competitiveness
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Thermotolerance – Ability to thrive at ≥ 37°C.
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Germination efficiency
Growth rate
Nutritional versatility – Ability to sense and
utilize nutrients in different forms and from
difference sources.
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Carbon
Nitrogen
Germination at 37°C
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Three most common
pathogens in
Aspergillus: fumigatus,
flavus, niger.
Prevalence as
pathogens correlates
with germination rate
at 37°C.
Araujo & Rodrigues. 2004.
J Clin Microbiol 42:4335.
Germination Rate
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Correlation is specific to
temperature, germination
rate at elevated temperature
is what correlates with
prevalence.
Organism must germinate
efficiently at body
temperature to have the
opportunity to be a
mammalian pathogen.
Araujo & Rodrigues. 2004.
J Clin Microbiol 42:4335.
Growth Rate Methods
Radial growth.
 Biomass.
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TOTAL 1 x104
CONIDIA
Turbidity.
 Dry weight.
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Not all methods give
the same answers.
MEASURE DIAMETER AT
24 AND 48 HOURS
RasB: Radial growth/Biomass and Virulence
110
100
90
80
80
70
% Survival
hyphal mass (mg)
100
60
60
40
50
wt
ΔrasB
rasB C'
20
1
0
0
growth rate (mm/hr)
0.8
2
4
6
8
10
Days
0.6
WT
0.4
0.2
0
wt
ΔrasB
ra sB C '
ΔrasB
rasB C'
12
14
Biomass (turbidity) and Virulence
Paisley, et al. 2005. Med Mycol 43:397.
CgrA: 37°C Radial Growth
Bhabhra, et al. 2004. Infect Immun 72:4731.
CgrA: Virulence
Mice
Bhabhra, et al. 2004. Infect Immun 72:4731.
Flies
ThtA: >37°C Growth & Virulence
thtA-
Chang, et al. 2004.
Fung Genet Biol 41:888.
Thermotolerance
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To be a mammalian pathogen, efficient
germination and good growth at 37°C are
required, but high temperature growth, >42°C
may not be.
Nutritional Versatility: Compost to Man
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A. fumigatus plays a key role in recycling C and N in compost.
Carbon sensing and utilization: pkaR and sakA.
Nitrogen sensing and utilization: rhbA, areA, cpcA, & sakA.
Auxotrophies: pabaA, pyrG, lysF.
PKA: Carbon Signaling and Growth
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In S. cerevisiae, mutants with hyperactive
cAMP/PKA signaling are unable to utilize nonfermentable carbon sources.
In A. fumigatus, ΔpkaR mutants are more growth
impaired on glycerol, than on glucose.
In A. fumigatus, PKA activity is high in the presence
of glucose, but low in the presence of glycerol.
Addition of cAMP to glycerol grown cultures of A.
fumigatus results in increased PKA activity.
Carbon Signaling – Regulation of alcA
Ethanol
Glucose
A
creA creA
C A
alcR
C
alcR
alcR alcR
alcA
A
C A
creA
alcA
C
creA
PkaR: C Sensing and Signaling
Ethanol
alcA
rRNA
WT DpkaR
- + - +
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In the wild type, alcA message
is induced over 10-fold in
response to ethanol, whereas in
the DpkaR strain, alcA message
was unchanged.
The lack of alcA induction may
indicate that carbon catabolite
repression is constitutively
engaged in the DpkaR strain.
Carbon sensing and/or
signaling is perturbed in ΔpkaR
mutant.
PkaR: Virulence
100
% Survival
80
60
WT
ΔpkaR
pkaR C'
40
20
0
0
2
4
6
8
Days
10
12
14
RhbA: Sensing Nitrogen Quality
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DrhbA
DrhbA + rhbA
)
rhbA
-1
RhbA functions
upstream in the TOR
growth and nutrient
sensing pathway.
RhbA responds to N
quality and quantity.
Radial Growth Rate (cm h
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0.08
*
0.07
0.06
*
**
HIS
PRO
0.05
0.04
0.03
0.02
0.01
0
NT
NO3
*p<0.05, **p<0.01
Panepinto, et al. 2003. Infect Immun 71:2819.
Regulation of rhbA: Nitrogen quantity
In vivo In vivo In vitro
24 h
72 h
24 h
rhbA 32.2 ±
9.6*
79.7 ±
22.5**
11.5 ±
4.2
Zhang, et al. 2005. Mycopathologia 160:201.
Panepinto, et al. 2002. Fung Genet Biol 36:207.
RhbA: Virulence
C
u
m
u
l
a
t
i
v
e
M
o
r
t
a
l
i
t
y
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2
0
1
5
Survial(no.fmice)
1
0
5
0
0
2
4
6
8
1
0
1
2
1
4
D
a
y
s
r
h
b
A
D
r
h
b
A
D
r
h
b
A
+
r
h
b
A
Panepinto, et al. 2003.
Infect Immun 71:2819.
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Virulence data
and in vivo
expression data
combine to
*suggest that “high
quality” N is not
readily available
in the host.
Counter-intuitive.
Auxotrophies and virulence
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Numerous auxotropies have been shown to
decrease virulence in A. fumigatus.
Suggests that some nutritional elements are in
short supply in the host.
LysF: Growth and Virulence
ΔlysF
ΔlysF
Liebman, et al. 2004.
Arch. Microbiol. 181:378.
PabaA: Virulence
PABA
stopped
Brown, et al. 2000. Mol Microbiol 36:4731.
How Did a Grass Eater Become an
Opportunistic Pathogen?
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Living in a compost pile translated into the
ability to:
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Germinate and grow efficiently at ≥ 37°C, i.e.,
thermotolerance.
Sense and utilize a variety of carbon and nitrogen
sources.
Make it’s own building block when necessary.
Make many conidia to compete in a hostile
environment.
Sometimes what makes a good grass eater can
also make a good opportunistic pathogen.
Acknowledgements
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Brian Oliver
John Panepinto
Jarrod Fortwendel
Wei Zhao
Tom Amlung
Darcey Smith
Amy Seitz
Lauren Fox
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David Askew
Doug Boettner
Ruchi Bhabhra
Mike Miley
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NIAID
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