|dc.description.abstract||Human systemic fungal infections are increasing, and causing high morbidity and mortality. Treatment is challenging because fungi share many metabolic pathways with mammals. Current antifungals are losing effectiveness due to drug resistance. In immunocompromised patients Aspergillus fumigatus causes systemic aspergillosis, the most important airborne fungal disease. Mortality from aspergillosis exceeds 50% even with aggressive treatment. We need novel antifungal drug targets. Fungal cell wall components are promising targets for antifungal therapy as they are essential for fungi and absent from humans.
The sugar galactofuranose (Galf) is a 5-memberd ring form of galactose that is found in the cell walls of many fungi, but not in mammals. I used molecular biology and microscopy techniques to characterize Galf biosynthesis enzymes in the model species A. nidulans. I studied three enzymes that catalyze sequential steps in Galf biosynthesis: UgmA, UgtA and UgeA. UDP-galactopyranose mutase (UgmA) creates UDP-galactofuranose (UDP-Galf) from UDP galactopyranose (UDP-Galp) in the cytoplasm. The UDP-Galf transporter (UgtA) moves UDP Galf into membrane bound organelles for incorporation into cell wall compartments. Upstream of UgmA, UDP-glucose/galactose epimerase (UgeA) interconverts UDP-glucose into UDP-Galp, the UgmA substrate. Neither UgmA nor UgtA has a human counterpart; UgeA is in the Leloir galactose metabolism pathway that found in many organisms from bacteria to humans.
None of UgeA, UgmA and UgtA is essential for viability of A. nidulans, but deleting any one of them substantially reduces colony growth and sporulation (Figure i). Wild type and Galf defective strains (ugeAΔ, ugmAΔ; and ugtAΔ) were quantified for colony growth, cell morphometry, spore formation and germination, as well as wall architecture. The abundance of these proteins was regulated using the alcA promoter. Galf content was assessed by immunolocalization in the Galf defective strains, showing that those strains lacked immunodetectable Galf. Gene products were localized with fluorescent protein tags; both UgmA and UgeA were cytoplasmic, whereas UgtA was Golgi localized. Wall surfaces were imaged and force-probed using transmission electron microscopy and atomic force microscopy. Overall, Galf deletion strains had aberrant wall maturation, and poorly consolidated surfaces. Our results indicate that Galf is necessary for abundant sporulation, wild type growth and full maturation of Aspergillus cell wall.
Galf deletion strains were assessed for sensitivity to antifungal agents in clinical use. They were significantly more sensitive to caspofungin and amphotericin B that target cell wall synthesis and cell membrane chemistry, respectively. Thus, anti-Galf drugs (once created) may be useful in combination with existing antifungal drugs. In summary, Galf biosynthesis pathway appears to be promising as an antifungal drug development target.||en_US