Aspergillus nidulans UDP-glucose-4-epimerase UgeA has multiple roles in wall architecture, hyphal morphogenesis, and asexual development Amira M. El-Ganiny a , Inder Sheoran b , David A.R. Sanders b , Susan G.W. Kaminskyj a, * a Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon SK, Canada S7N 5E2 b Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon SK, Canada S7N 5C9 article info Article history: Received 21 January 2010 Accepted 2 March 2010 Available online 6 March 2010 Keywords: Aspergillus nidulans Galactofuranose Hyphal morphogenesis Hyphal wall architecture UDP-glucose-4-epimerase Drug development target abstract Aspergillus nidulans UDP-glucose-4-epimerase UgeA interconverts UDP-glucose and UDP-galactose and participates in galactose metabolism. The sugar moiety of UDP-galactose is predominantly found as galactopyranose (Galp, the six-membered ring form), which is the substrate for UDP-galactopyranose mutase (encoded by ugmA) to generate UDP-galactofuranose (Galf, the five-membered ring form) that is found in fungal walls. In A. fumigatus, Galf residues appear to be important for virulence. The A. nidulans ugeAD strain is viable, and has defects including wide, slow growing, highly branched hyphae and reduced conidiation that resemble the ugmAD strain. As for the ugmAD strain, ugeAD colonies had sub- stantially reduced sporulation but normal spore viability. Conidia of the ugeAD strain could not form col- onies on galactose as a sole carbon source, however they produced short, multinucleate germlings suggesting they ceased to grow from starvation. UgeA purified from an expression plasmid had a relative molecular weight of 40.6 kDa, and showed in vitro UDP-glucose-4-epimerase activity. Transmission elec- tron microscope cross-sections of wildtype, ugeAD, and ugmAD hyphae showed they had similar cyto- plasmic contents but the walls of each strain were different in appearance and thickness. Both deletion strains showed increased substrate adhesion. Localization of UgeA-GFP and UgmA-GFP was cytoplasmic, and was similar on glucose and galactose. Neither gene product had a longitudinal polarized distribution. Localization of a UgmA-mRFP in a strain that resembled the ugmAD strain was cytoplasmic and lacked a longitudinal polarized distribution. The roles of UgeA in A. nidulans growth and morphogen- esis are consistent with the importance of Galf, and are related but not identical to the roles of UgmA. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction Fungi and protozoa are morphologically simple eukaryotes that are becoming increasingly important human pathogens. In 1939, Aspergillus fumigatus infections were considered to be ‘so rare as to be of little practical importance’ (reviewed in Latgé and Stein- bach (2009)), whereas now A. fumigatus is considered to be a pre- dominant opportunistic and also a primary human fungal pathogen (Fedorova et al., 2008). Diseases caused by fungi and pro- tozoa are often therapeutically intractable due to their underlying metabolic similarities with animal systems, and systemic fungal infections have high mortality even with aggressive treatment. There are relatively few antifungal drugs, most targeting ergosterol and its biosynthetic pathway (amphotericin B, azoles, allylamines) or more recently against fungal cell walls (echinocandins), but many of these are losing effectiveness due emerging fungal resis- tance (Cowen, 2008). About 20% of Saccharomyces cerevisiae (Lesage and Bussey, 2006) and 12% of Aspergillus nidulans (Harris et al., 2009) gene function relates to cell wall formation and maintenance. The fungal cell wall comprises about 20% of its biomass. The fungal wall medi- ates interactions between the fungal organism and its environ- ment, which is the host in the case of pathogenic species (Klis et al., 2007). Wall function is generally resilient to deletion of indi- vidual genes (de Groot et al., 2009) and to chemical stressors that trigger compensatory cell wall stress response pathways (Damveld et al., 2008). Fungal walls are remodeled during growth in culture (Momany et al., 2004) and in response to their environment (Gastebois et al., 2009; Hurtado-Guerrero et al., 2009). As with other human fungal pathogens, A. fumigatus forms biofilms in host tissue (Seidler et al., 2008; Loussert et al., 2010) that are more drug resistant than planktonic cultures (Seidler et al., 2008). Since fun- gal extracellular carbohydrates are not found in animal systems (Beverly et al., 2005), they and their biosynthetic pathways are drug development targets (Pederson and Turco, 2003). 1087-1845/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2010.03.002 Abbreviations: CLSM, confocal laser scanning microscopy; Galf, galactofuranose; Galp, galactopyranose; GFP, green fluorescent protein; mRFP, monomeric red fluorescent protein; SEM, scanning electron microscopy; TEM, transmission elec- tron microscopy; UGE, UDP-glucose-4-epimerase; UGM, UDP-galactopyranose mutase. * Corresponding author. Fax: +1 306 966 4461. E-mail address: susan.kaminskyj@usask.ca (S.G.W. Kaminskyj). Fungal Genetics and Biology 47 (2010) 629–635 Contents lists available at ScienceDirect Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi