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

2. Aspergillus fumigatus Ubiquitous organism. Most commonly reported opportunistic hyphomycete. Important in compost cycle. How did this grass eater become an opportunistic pathogen?

3. 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?

4. 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

5. 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.

6. 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.

7. Growth Rate Methods Radial growth. Biomass. Turbidity. Dry weight. Not all methods give the same answers. TOTAL 1 x10 4 CONIDIA MEASURE DIAMETER AT 24 AND 48 HOURS

8. RasB: Radial growth/Biomass and Virulence

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

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

11. CgrA: Virulence Mice Flies Bhabhra, et al. 2004. Infect Immun 72:4731.

12. ThtA: >37°C Growth & Virulence Chang, et al. 2004. Fung Genet Biol 41:888. thtA-

13. 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.

14. 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.

15. 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.

16. Carbon Signaling – Regulation of alcA alcA A C A C creA alcR creA alcA A C A C alcR Glucose Ethanol

17. 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

18. PkaR: Virulence

19. RhbA: Sensing Nitrogen Quality RhbA functions upstream in the TOR growth and nutrient sensing pathway. RhbA responds to N quality and quantity. rhbA rhbA + rhbA *p<0.05, **p<0.01 Panepinto, et al. 2003. Infect Immun 71:2819.

20. 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.

21. 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.

22. 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.

23. LysF: Growth and Virulence Liebman, et al. 2004. Arch. Microbiol. 181:378. ΔlysF

24. PabaA: Virulence PABA stopped Brown, et al. 2000. Mol Microbiol 36:4731.

25. 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 its 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.

26. 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