Aspergillus fumigatus Growth and Virulence Judith C Rhodes

Aspergillus fumigatus: Growth and Virulence Judith C. Rhodes, Ph. D. University of Cincinnati, OH, USA judith. rhodes@uc. edu

Aspergillus fumigatus o o 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 o o 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 o Thermotolerance – Ability to thrive at ≥ 37°C. n n o Germination efficiency Growth rate Nutritional versatility – Ability to sense and utilize nutrients in different forms and from difference sources. n n Carbon Nitrogen

Germination at 37°C o o 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 o o 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 o Radial growth. o Biomass. TOTAL 1 x 104 CONIDIA Turbidity. n Dry weight. n o Not all methods give the same answers. MEASURE DIAMETER AT 24 AND 48 HOURS

Ras. B: Radial growth/Biomass and Virulence

Biomass (turbidity) and Virulence Paisley, et al. 2005. Med Mycol 43: 397.

Cgr. A: 37°C Radial Growth Bhabhra, et al. 2004. Infect Immun 72: 4731.

Cgr. A: Virulence Mice Bhabhra, et al. 2004. Infect Immun 72: 4731. Flies

Tht. A: >37°C Growth & Virulence tht. A- Chang, et al. 2004. Fung Genet Biol 41: 888.

Thermotolerance o 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 o o A. fumigatus plays a key role in recycling C and N in compost. Carbon sensing and utilization: pka. R and sak. A. Nitrogen sensing and utilization: rhb. A, are. A, cpc. A, & sak. A. Auxotrophies: paba. A, pyr. G, lys. F.

PKA: Carbon Signaling and Growth o o In S. cerevisiae, mutants with hyperactive c. AMP/PKA signaling are unable to utilize nonfermentable carbon sources. In A. fumigatus, Δpka. R 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 c. AMP to glycerol grown cultures of A. fumigatus results in increased PKA activity.

Carbon Signaling – Regulation of alc. A Ethanol Glucose A cre. A C A alc. R C alc. R alc. A alc. R A C A cre. A alc. A C cre. A

Pka. R: C Sensing and Signaling Ethanol alc. A r. RNA WT Dpka. R - + o o o In the wild type, alc. A message is induced over 10 -fold in response to ethanol, whereas in the Dpka. R strain, alc. A message was unchanged. The lack of alc. A induction may indicate that carbon catabolite repression is constitutively engaged in the Dpka. R strain. Carbon sensing and/or signaling is perturbed in Δpka. R mutant.

Pka. R: Virulence

Rhb. A: Sensing Nitrogen Quality o o Rhb. A functions upstream in the TOR growth and nutrient sensing pathway. Rhb. A responds to N quality and quantity. rhb. A Drhb. A + rhb. A *p<0. 05, **p<0. 01 Panepinto, et al. 2003. Infect Immun 71: 2819.

Regulation of rhb. A: Nitrogen quantity In vivo In vitro 24 h 72 h 24 h rhb. A 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.

Rhb. A: Virulence o o Panepinto, et al. 2003. Infect Immun 71: 2819. 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 o o Numerous auxotropies have been shown to decrease virulence in A. fumigatus. Suggests that some nutritional elements are in short supply in the host.

Lys. F: Growth and Virulence Δlys. F Liebman, et al. 2004. Arch. Microbiol. 181: 378.

Paba. A: Virulence PABA stopped Brown, et al. 2000. Mol Microbiol 36: 4731.

How Did a Grass Eater Become an Opportunistic Pathogen? o Living in a compost pile translated into the ability to: n n o 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 o o o o Brian Oliver John Panepinto Jarrod Fortwendel Wei Zhao Tom Amlung Darcey Smith Amy Seitz Lauren Fox o David Askew Doug Boettner Ruchi Bhabhra Mike Miley o NIAID o o o