Metabolism and morphogenesis of human pathogenic fungi
Final Report Abstract
This project generated the basis for elucidating the importance of lysine biosynthesis for establishment of invasive aspergillosis caused by the filamentous ascomycete "Aspergillus fumigatus". For this purpose the homocitrate synthase from "A. fumigatus" was biochemically characterised and found to be highly specific for the substrates acetyl-CoA and 2-oxoglutarate. A gene deletion resulted in lysine auxotrophic strains, confirming that no other enzyme can substitute for the homocitrate synthase gene. Subsequent virulence studies revealed that the homocitrate synthase mutant was strongly attenuated in a pulmonary aspergillosis model, implying that the pathway may indeed act as a possible target for novel antifungal drugs. However, the mutant showed normal virulence in a disseminated bloodstream infection model and further analyses revealed that the lysine support within the bloodstream is sufficient for conidia germination. Therefore, drugs targeting lysine biosynthesis may only be useful for prophylactic therapy in high risk patients, but not suitable for a therapeutic approach of established disease. While the alpha-aminoadipate pathway is essential in fungi for lysine biosynthesis, it also provides an essential precursor in beta-lactam antibiotics synthesis. While most enzymes involved in the alpha-aminoadipate pathway were already known for decades, the isomerisation of homocitrate to homoisocitrate via homoaconitate had hardly been investigated. Here, it had been believed that the homoaconitase performs the complete isomerisation. However, we discovered that the homoaconitase only catalyses the second half reaction, which is the reversible hydration of homoaconitate to homoisocitrate. We subsequently identified the citric acid cycle aconitase as essential contributor in this isomerisation by catalysing the first half-reaction. Thus, citric acid cycle and lysine biosynthesis are tightly linked in fungi. Interestingly, the yeast "Saccharomyces cerevisiae" unlinked both pathways by expressing a second aconitase that had specifically adapted to lysine biosynthesis. Taking the fermentative life-style of yeasts into account, this uncoupling may have an evolutionary advantage. On the contrary, filamentous fungi rely on the citric acid cycle and therefore need to operate both pathways in parallel.
Publications
- 2010. Evaluation of lysine biosynthesis as antifungal drug target: Biochemical characterisation of Aspergillus fumigatus homocitrate synthase and virulence studies. Eukaryotic Cell, Vol. 9. 2010, no. 6, pp. 878-893.
Schöbel F, Jacobsen ID, Brock M
(See online at https://dx.doi.org/10.1128/EC.00020-10) - 2012. The fungal α-aminoadipate pathway for lysine biosynthesis requires two enzymes of the aconitase family for the isomerisation of homocitrate to homoisocitrate. Molecular Microbiology, Vol. 86. 2012, Issue 6, pp. 1508–1530.
Fazius F, Shelest E, Gebhardt P, Brock M
(See online at https://doi.org/10.1111/mmi.12076) - 2013. Lysine biosynthesis in microbes: relevance as drug target and prospects for β-lactam antibiotic production. Applied Microbiology and Biotechnology, Vol. 97. 2013, Issue 9, pp 3763-3772.
Fazius F, Zaehle C, Brock M
(See online at https://doi.org/10.1007/s00253-013-4805-1)
