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Aspergillus fumigatus:Growth and VirulenceJudith C. Rhodes, Ph.D.
University of Cincinnati
Cincinnati, OH, USA
Aspergillus fumigatus 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
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
Thermotolerance – Ability to thrive at ≥ 37°C. Germination efficiency Growth rate
Nutritional versatility – Ability to sense and utilize nutrients in different forms and from difference sources. Carbon Nitrogen
Germination at 37°C
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 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.
Turbidity. Dry weight.
Not all methods give the same answers.
TOTAL 1 x104
CONIDIA
MEASURE DIAMETER AT 24 AND 48 HOURS
RasB: Radial growth/Biomass and Virulence
0
20
40
60
80
100
0 2 4 6 8 10 12 14
Days
% S
urv
ival
WT ΔrasB rasB C'
0
0.2
0.4
0.6
0.8
1
wt ΔrasB rasB C'
gro
wth
rat
e (m
m/h
r)
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 Flies
Bhabhra, et al. 2004. Infect Immun 72:4731.
ThtA: >37°C Growth & Virulence
Chang, et al. 2004. Fung Genet Biol 41:888.
thtA-
Thermotolerance
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
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
In S. cerevisiae, mutants with hyperactive cAMP/PKA signaling are unable to utilize non-fermentable 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
alcAA C A CcreA creA
alcRalcR creA
creA
alcAA C A CalcRalcR
Glucose Ethanol
PkaR: C Sensing and Signaling
In the wild type, alcA message is induced over 10-fold in response to ethanol, whereas in the pkaR strain, alcA message was unchanged.
The lack of alcA induction may indicate that carbon catabolite repression is constitutively engaged in the pkaR strain.
Carbon sensing and/or signaling is perturbed in ΔpkaR mutant.
WT pkaR
- -+ +
alcA
rRNA
Ethanol
PkaR: Virulence
0
20
40
60
80
100
0 2 4 6 8 10 12 14
Days
% S
urv
ival WT
ΔpkaR
pkaR C'
RhbA: Sensing Nitrogen Quality RhbA: Sensing Nitrogen Quality
RhbA functions upstream in the TOR growth and nutrient sensing pathway.
RhbA responds to N quality and quantity. 0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
NT NO3 HIS PRO
Ra
dia
l G
row
th R
ate
(c
m h
-1)
* * **
rhbA rhbA rhbA + rhbA
*p<0.05, **p<0.01
Panepinto, et al. 2003. Infect Immun 71:2819.
Regulation of rhbA: Nitrogen quantity
In vivo
24 h
In vivo
72 h
In vitro
24 h
rhbA 32.2 ±
9.6*
79.7 ±
22.5**
11.5 ±
4.2
Panepinto, et al. 2002. Fung Genet Biol 36:207.
Zhang, et al. 2005. Mycopathologia 160:201.
RhbA: Virulence Virulence data
and in vivo expression data combine to suggest that “high quality” N is not readily available in the host.
Counter-intuitive.
*
Panepinto, et al. 2003.Infect Immun 71:2819.
Auxotrophies and virulence
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
Liebman, et al. 2004. Arch. Microbiol. 181:378.
ΔlysF
ΔlysF
PabaA: VirulencePABA stopped
Brown, et al. 2000. Mol Microbiol 36:4731.
How Did a Grass Eater Become an Opportunistic Pathogen? Living in a compost pile translated into the
ability to: 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 Brian Oliver John Panepinto Jarrod Fortwendel Wei Zhao Tom Amlung Darcey Smith Amy Seitz Lauren Fox
David Askew Doug Boettner Ruchi Bhabhra Mike Miley
NIAID
