Sialic acid metabolism in the opportunistic fungal pathogen, Aspergillus fumigatus

My research investigated sialic acid metabolism in the opportunistic fungal pathogen, Aspergillus fumigatus. The sialic acid, N-acetylneuraminic acid (Neu5Ac), is a sugar found on fungal spore cell surface that mediates adhesion to host proteins and phagocytes. The aims of the thesis were to characterize a novel A. fumigatus exo-sialidase (AfS), and to clone and characterize putative A. fumigatus nucleotide sugar transporters (AfNSTs) to identify CMP-Neu5Ac or UDP-galactose transporters. The A. fumigatus sialidase gene was expressed in E. coli and crystallized; the crystal structure and Michaelis – Menten kinetic analysis revealed that the glycoside of another sialic acid, 2-keto-3-deoxynononic acid (KDN), was a better substrate for the enzyme than glycosides of Neu5Ac. This enzyme represents the first sialidase characterized from the Kingdom Fungi. To better understand why KDN is a better substrate for AfS than Neu5Ac, using the enzyme structure as a guide in conjunction with known sialidase structures, a point-mutation (R151L) was introduced in the substrate binding pocket to better accommodate glycans with terminal Neu5Ac. Activity of the R151L mutant was slightly enhanced toward Neu5Ac. Moreover, amino acid sequence comparisons revealed that this amino acid may be a hallmark of KDNases. In addition, I attempted to identify a CMP-sialic acid transporter in A. fumigatus, a type of nucleotide sugar transporter (NST). NSTs mediate nucleotide sugar transport into the endoplasmic reticulum and Golgi complex for subsequent addition to glycoproteins and glycolipids. STD-NMR analysis and 14C-transport assays were conducted to examine the substrate specificity of four putative A. fumigatus NSTs expressed in yeast. Two transporters (AfNST1 and AfNST5) bound UDP-glucose and UDP-galactose, and transported 14C-UDP-galactose. Epitope maps showed that the UDP-moiety anchored the nucleotide sugar and that sugar structure conferred specificity because not all UDP-sugars bound to the NSTs. No CMP-sialic acid transport was detected. Despite similarities in substrate preference between AfNST1 and AfNST5, growth and morphology of the corresponding knock-out mutants differed; only the Af∆NST5KO was compromised when grown on media containing cell wall stressors. Using lectins and flow cytometry, I found that the level of cell surface galactose was significantly reduced in both knockout strains as compared to the wild type; however, sialic acid density on conidia was significantly reduced only in the Af∆NST5KO mutant. This research demonstrates for the first time that NSTs are important for the integrity of the fungal cell and may represent novel targets for antifungal agents.