Search

Glycoinositolphosphoceramides (GIPCs) are complex sphingolipids present at the plasma membrane of various eukaryotes with the important exception of mammals. In fungi, these glycosphingolipids commonly contain an a-mannose residue (Man) linked at position 2 of the inositol. However, several pathogenic fungi additionally synthesize zwitterionic GIPCs carrying an a-glucosamine residue (GlcN) at this position. In the human pathogen Aspergillus fumigatus, the GlcNα1,2IPC core (where IPC is inositolphosphoceramide) is elongated to Manα1,3Manα1,6GlαNα1,2IPC, which is the most abundant GIPC synthesized by this fungus. In this study, we identified an A. fumigatus Nacetylglucosaminyltransferase, named GntA, and demonstrate its involvement in the initiation of zwitterionic GIPC biosynthesis. Targeted deletion of the gene encoding GntA in A. fumigatus resulted in complete absence of zwitterionic GIPC; a phenotype that could be reverted by episomal expression of GntA in themutant. The N-acetylhexosaminyltransferase activity of GntAwas substantiated by production of N-acetylhexosamine-IPC in the yeast Saccharomyces cerevisiae upon GntA expression. Using an in vitro assay, GntAwas furthermore shown to use UDP-N-acetylglucosamine as donor substrate to generate a glycolipid product resistant to saponification and to digestion by phosphatidylinositol- phospholipase C as expected for GlcNAca1,2IPC. Finally, as the enzymes involved in mannosylation of IPC, GntA was localized to the Golgi apparatus, the site of IPC synthesis. [ABSTRACT FROM AUTHOR]

Fungal cell walls frequently contain a polymer of mannose and galactose called galactomannan. In the pathogenic filamentous fungus Aspergillus fumigatus, this polysaccharide is made of a linear mannan backbone with side chains of galactofuran and is anchored to the plasma membrane via a glycosylphosphatidylinositol or is covalently linked to the cell wall. To date, the biosynthesis and significance of this polysaccharide are unknown. The present data demonstrate that deletion of the Golgi UDP-galactofuranose transporter GlfB or the GDP-mannose transporter GmtA leads to the absence of galactofuran or galactomannan, respectively. This indicates that the biosynthesis of galactomannan probably occurs in the lumen of the Golgi apparatus and thus contrasts with the biosynthesis of other fungal cell wall polysaccharides studied to date that takes place at the plasma membrane. Transglycosylation of galactomannan from the membrane to the cell wall is hypothesized because both the cell wall-bound and membrane-bound polysaccharide forms are affected in the generated mutants. Considering the severe growth defect of the A. fumigatus GmtA-deficient mutant, proving this paradigm might provide new targets for antifungal therapy. [ABSTRACT FROM AUTHOR]