Structural and biochemical studies on the mechanism of CO2-sensing in the virulence of pathogenic fungi
Final Report Abstract
Carbon dioxide (CO2) can act as a signalling molecule and, e.g. it enables mosquitos to find their prey. In the human-pathogenic fungi Candida albicans and Cryptococcus neoformans, a CO2-sensing system regulates the switch from growth in a natural environment (~0.03 % CO2) to virulent growth in the human host (~5 % CO2). This system comprises the fungal adenylyl cyclase, which is stimulated by bicarbonate, and a carbonic anhydrase which forms this bicarbonate. Knocking out either enzyme prevents fungal pathogenesis under certain conditions suggesting them as targets for anti-fungal drugs. We studied this signalling system in Candida albicans and Cryptococcus neoformans by using biochemical methods and protein crystallography. Can2 is the carbonic anhydrase of the CO2-sensing system in C. neoformans, and Nce103 the one in C. albicans. We crystallized Can2 and solved its structure at 1.7 Å resolution using Patterson search techniques with the structure of a homolog from E. coli as search model. The Can2 structure reveals that the enzyme belongs to the "plant-type" beta-CAs but features a unique N-terminal extension. This extension interacts with the active-site entrance of the dimeric enzyme, suggesting a regulatory function. By testing a panel of compounds with a collaborator, nanomolar Can2 inhibitors could be identified, and by solving the structure of a Can2 complex with the product analog acetate and modeling of Can2 complexes with inhibitors, we could obtain insights into the interplay of substrate, inhibitor, and the regulatory N-terminal extension. The inhibitors were shown by a collaborator to prevent pathogenic growth, albeit at elevated concentrations so that further improvement will be required for development of a therapeutic drug. The crystal structures were also used for rationalizing further inhibitors identified in screens conducted with our recombinant enzyme, identifying e.g. cyanide and carbonate as potent zinc-binding groups (ZBGs) selectively for Nce103 and sulfonamides such as acetazolamide and 4-hydroxymethylbenzenesulfonamide as nanomolar inhibitors. A homology model for Nce103 based on the crystal structure of Can2 revealed that ZBGs with less polar moieties and compact scaffolds generate stronger Nce103 inhibitors, whereas more polar ZBGs with bulkier moieties are more promising for Can2 inhibition. These results thus provide compounds and structural information that improve our understanding of the carbonic anhydrases involved in CO2-sensing and that support development of drugs targeting this system. We further studied the role of the adenylyl cyclases Cyr1p from C. albicans in this CO2-sensing system by generating and analyzing a homology model of this enzyme. We thereby designed a system that provides Cyr1p activity but knocks out its bicarbonate-dependent activation by removing a key residue for bicarbonate binding. Using this system, our collaborators could then show in fungal cultures that Cyr1p acts as physiological CO2-sensor in determining filamentous growth, and in an in vivo model that this sensor function is relevant for inducing pathogenicity in a fly-based infection model. These results confirm the CO2-sensing function of a system comprising Nce103 and Cyr1p in fungal growth and pathogenicity and identifies its molecular mechanism comprising bicarbonate formation and its specific binding to an adenylyl cyclases active site pocket. These insights improve our understanding of a metabolic and environmental sensing system and support the development of drugs targeting this system.
Publications
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Carbonic anhydrase inhibitors. Inhibition of the beta-class enzymes from the fungal pathogens Candida albicans and Cryptococcus neoformans with simple anions. Bioorganic & Medicinal Chemistry Letters, Vol. 18. 2008, Issue 18, pp. 5066–5070.
A. Innocenti, F.A. Mühlschlegel, R.A. Hall, C. Steegborn, A. Scozzafava, C.T. Supuran
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Carbonic anhydrase inhibitors. Inhibition and homology modeling studies of the fungal beta-carbonic anhydrase from Candida albicans with sulfonamides. Bioorganic & Medicinal Chemistry, Vol. 17. 2009, Issue 13, pp. 4503–4509.
A. Innocenti, R.A. Hall, C. Schlicker, A. Scozzafava, C. Steegborn, F.A. Mühlschlegel, C.T. Supuran
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Crystallographic studies on carbonic anhydrases from fungal pathogens for structure-assisted drug development. In: C.T. Supuran, J.-Y. Winum (Eds) Drug Design of Zinc-Enzyme Inhibitors: Functional, Structural, and Disease Applications. 2009, pp. 323–333.
U.-M. Ohndorf, C. Schlicker, C. Steegborn
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Structure and inhibition of the CO2-sensing carbonic anhydrase Can2 from the pathogenic fungus Cryptococcus neoformans. Journal of Molecular Biology, Vol. 385. 2009, Issue 4, Pages 1207–1220.
C. Schlicker, R.A. Hall, D. Vullo, S. Middelhaufe, M. Gertz, C.T. Supuran, F.A. Mühlschlegel, C. Steegborn
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CO2 Acts as a Signalling Molecule in Populations of the Fungal Pathogen Candida albicans. PLoS Pathogens 6, e1001193.
A. Hall, L. De Sordi, D. M. MacCallum, H. Topal, R. Eaton, J. W. Bloor, G. K. Robinson, L. R. Levin, J. Buck, Y. Wang, N. A. R. Gow, C. Steegborn, F. A. Mühlschlegel
