Dataset Information

A single UDP-galactofuranose transporter is required for galactofuranosylation in Aspergillus fumigatus.

ABSTRACT: Galactofuranose (Galf) containing molecules have been described at the cell surface of several eukaryotes and shown to contribute to the virulence of the parasite Leishmania major and the fungus Aspergillus fumigatus. It is anticipated that a number of the surface glycoconjugates such as N-glycans or glycolipids are galactofuranosylated in the Golgi apparatus. This raises the question of how the substrate for galactofuranosylation reactions, UDP-Galf, which is synthesized in the cytosol, translocates into the organelles of the secretory pathway. Here we report the first identification of a Golgi-localized nucleotide sugar transporter, named GlfB, with specificity for a UDP-Galf. In vitro transport assays established binding of UDP-Galf to GlfB and excluded transport of several other nucleotide sugars. Furthermore, the implication of glfB in the galactofuranosylation of A. fumigatus glycoconjugates and galactomannan was demonstrated by a targeted gene deletion approach. Our data reveal a direct connection between galactomannan and the organelles of the secretory pathway that strongly suggests that the cell wall-bound polysaccharide originates from its glycosylphosphatidylinositol-anchored form.

SUBMITTER: Engel J

PROVIDER: S-EPMC2797156 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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A single UDP-galactofuranose transporter is required for galactofuranosylation in Aspergillus fumigatus.

Engel Jakob J Schmalhorst Philipp S PS Dörk-Bousset Thilo T Ferrières Vincent V Routier Françoise H FH

The Journal of biological chemistry 20091019 49

Galactofuranose (Galf) containing molecules have been described at the cell surface of several eukaryotes and shown to contribute to the virulence of the parasite Leishmania major and the fungus Aspergillus fumigatus. It is anticipated that a number of the surface glycoconjugates such as N-glycans or glycolipids are galactofuranosylated in the Golgi apparatus. This raises the question of how the substrate for galactofuranosylation reactions, UDP-Galf, which is synthesized in the cytosol, translo ...[more]

PMID: 19840949

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Project description:BackgroundGalactofuranose (Galf)-containing glycoconjugates are important to secure the integrity of the cell wall of filamentous fungi. Mutations that prevent the biosynthesis of Galf-containing molecules compromise cell wall integrity. In response to cell wall weakening, the cell wall integrity (CWI)-pathway is activated to reinforce the strength of the cell wall. Activation of CWI-pathway in Aspergillus niger is characterized by the specific induction of the agsA gene, which encodes a cell wall α-glucan synthase.ResultsIn this study, we screened a collection of cell wall mutants with an induced expression of agsA for defects in Galf biosynthesis using a with anti-Galf antibody (L10). From this collection of mutants, we previously identified mutants in the UDP-galactopyranose mutase encoding gene (ugmA). Here, we have identified six additional UDP-galactopyranose mutase (ugmA) mutants and one mutant (named mutant #41) in an additional complementation group that displayed strongly reduced Galf-levels in the cell wall. By using a whole genome sequencing approach, 21 SNPs in coding regions were identified between mutant #41 and its parental strain which changed the amino acid sequence of the encoded proteins. One of these mutations was in gene An14g03820, which codes for a putative UDP-glucose-4-epimerase (UgeA). The A to G mutation in this gene causes an amino acid change of Asn to Asp at position 191 in the UgeA protein. Targeted deletion of ugeA resulted in an even more severe reduction of Galf in N-linked glucans, indicating that the UgeA protein in mutant #41 is partially active. The ugeA gene is also required for growth on galactose despite the presence of two UgeA homologs in the A. niger genome.ConclusionBy using a classical mutant screen and whole genome sequencing of a new Galf-deficient mutant, the UDP-glucose-4-epimerase gene (ugeA) has been identified. UgeA is required for the biosynthesis of Galf as well as for galactose metabolism in Aspergillus niger.

Project description:The number of patients suffering from fungal diseases has constantly increased during the last decade. Among the fungal pathogens, the airborne filamentous fungus Aspergillus fumigatus can cause chronic and fatal invasive mold infections. So far, only three major classes of drugs (polyenes, azoles, and echinocandins) are available for the treatment of life-threatening fungal infections, and all present pharmacological drawbacks (e.g., low solubility or toxicity). Meanwhile, clinical antifungal-resistant isolates are continuously emerging. Therefore, there is a high demand for novel antifungal drugs, preferentially those that act on new targets. We studied urease and the accessory proteins in A. fumigatus to determine their biochemical roles and their influence on virulence. Urease is crucial for the growth on urea as the sole nitrogen source, and the transcript and protein levels are elevated on urea media. The urease deficient mutant displays attenuated virulence, and its spores are more susceptible to macrophage-mediated killing. We demonstrated that this observation is associated with an inability to prevent the acidification of the phagosome. Furthermore, we could show that a nickel-chelator inhibits growth on urea. The nickel chelator is also able to reverse the effects of urease on macrophage killing and phagosome acidification, thereby reducing virulence in systemic and trachea infection models. IMPORTANCE The development of antifungal drugs is an urgent task, but it has proven to be difficult due to many similarities between fungal and animal cells. Here, we characterized the urease system in A. fumigatus, which depends on nickel for activity. Notably, nickel is not a crucial element for humans. Therefore, we went further to explore the role of nickel-dependent urease in host-pathogen interactions. We were able to show that urease is important in preventing the acidification of the phagosome and therefore reduces the killing of conidia by macrophages. Furthermore, the deletion of urease shows reduced virulence in murine infection models. Taken together, we identified urease as an essential virulence factor of A. fumigatus. We were able to show that the application of the nickel-chelator dimethylglyoxime is effective in both in vitro and in vivo infection models. This suggests that nickel chelators or urease inhibitors are potential candidates for the development of novel antifungal drugs.

Dataset Information

A single UDP-galactofuranose transporter is required for galactofuranosylation in Aspergillus fumigatus.

Publications

A single UDP-galactofuranose transporter is required for galactofuranosylation in Aspergillus fumigatus.

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