Biosynthesis of glucosamine containing glycolipids in the fungal pathogen Aspergillus fumigatus: New enzmes, new drug targets?
Zusammenfassung der Projektergebnisse
The vast majority of eukaryotic gene clusters have been found to encode secondary metabolites in fungi such as antibiotics, immunosuppressants or poisons. In this project, we described a gene cluster of the human pathogen Aspergillus fumigatus and demonstrated its involvement in the biosynthesis of the atypical and abundant zwitterionic glycolipid Af3c (Manα1,3Manα1,6GlcNα1,2IPC, where Man is mannose; GlcN is glucosamine and IPC is inositolphosphoceramide). The four enzymes encoded by the gene cluster, namely an α1,2-N-acetylglucosaminyltransferase (GntA), a de-N-acetylase (GdaA), an α1,6-mannosyltransferase (OchC) and α1,3-mannosyltransferase (ClpC), were functionally characterised by reconstitution of the Af3c glycolipid biosynthetic pathway in the yeast Saccharomyces cerevisiae. Moreover, absence of zwitterionic glycolipid in A. fumigatus after targeted deletion of the gene encoding GntA substantiated the role of this biosynthetic gene cluster. We furthermore demonstrated that GntA is an UDP-GlcNAc:IPC α1,2-N-acetylglucosaminyltransferase acting in the Golgi lumen and is dispensable for fungal growth in vitro. Examination of sequenced fungal genomes revealed the presence of the Af3c gene cluster or of a sub-cluster in a few saprotrophic ascomycetes. The genes organization and distribution suggest that the zwitterionic glycolipid Af3c is an allelochemical enhancing survival of Aspergillus fumigatus in its natural habitat, thereby contributing to its environmental abundance. If the zwitterionic glycolipid Af3c provides an advantage for invasion and growth in the lungs remains to be confirmed. In the second part of this project, we examined the A. fumigatus CAP59 protein family comprising ClpA, ClpB and the α1,3-mannosyltransferase ClpC acting in Af3c biosynthesis. In the pathogenic fungus Cryptococcus neoformans, CAP59 has been involved in capsule formation and virulence but its precise function remains elusive. Our analysis revealed that like ClpC, ClpB may act in the biosynthesis of acidic and zwitterionic glycolipids. Moreover, using various techniques, we demonstrated that ClpA is a Golgi localized α1,3- mannosyltransferase modifying the fungal GPI-anchor core to generate the structure Manα1,3Manα1,2Manα1,2Manα1,6 Manα1,4GlcNα1,6IPC. Deletion of clpA in A. fumigatus resulted in the quasi absence of carbohydrate modification of the fungal GPI core and provides a system to determine if these carbohydrate “decorations” may affect transfer of GPI-anchored proteins to the cell wall.
Projektbezogene Publikationen (Auswahl)
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(2015). Aspergillus Fumigatus CAP59 Like Protein A is involved in α1,3-Mannosylation of GPI-Anchors. Glycobiology
Krüger A.T., Engel J., Buettner F.F.R., Routier F.H.
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(2015). Characterisation of an N-Acetylglucosaminyltransferase involved in Aspergillus Fumigatus Zwitterionic Glycoinositolphosphoceramide Biosynthesis. Glycobiology
Engel J, Schmalhorst P.S., Krüger A.T., Müller C.T., Buettner F.F.R., Routier F.H.
