Abstract
BackgroundGalactofuranose (Galf)-containing glycoconjugates are present in numerous microbes, including filamentous fungi where they are important for morphology, virulence and maintaining cell wall integrity. The incorporation of Galf-residues into galactomannan, galactomannoproteins and glycolipids is carried out by Golgi-localized Galf transferases. The nucleotide sugar donor used by these transferases (UDP-Galf) is produced in the cytoplasm and has to be transported to the lumen of the Golgi by a dedicated nucleotide sugar transporter.MethodsBased on homology with recently identified UDP-Galf-transporters in A. fumigatus and A. nidulans, two putative UDP-Galf-transporters in A. niger were found. Their function and localization was determined by gene deletions and GFP-tagging studies, respectively.ResultsThe two putative UDP-Galf-transporters in A. niger are homologous to each other and are predicted to contain eleven transmembrane domains (UgtA) or ten transmembrane domains (UgtB) due to a reduced length of the C-terminal part of the UgtB protein. The presence of two putative UDP-Galf-transporters in the genome was not unique for A. niger. From the twenty Aspergillus species analysed, nine species contained two additional putative UDP-Galf-transporters. Three of the nine species were outside the Aspergillus section nigri, indication an early duplication of UDP-Galf-transporters and subsequent loss of the UgtB copy in several aspergilli. Deletion analysis of the single and double mutants in A. niger indicated that the two putative UDP-Galf-transporters (named UgtA and UgtB) have a redundant function in UDP-Galf-transport as only the double mutant displayed a Galf-negative phenotype. The Galf-negative phenotype of the double mutant could be complemented by expressing either CFP-UgtA or CFP-UgtB fusion proteins from their endogenous promoters, indicating that both CFP-tagged proteins are functional. Both Ugt proteins co-localize with each other as well as with the GDP-mannose nucleotide transporter, as was demonstrated by fluorescence microscopy, thereby confirming their predicted localization in the Golgi.ConclusionA. niger contains two genes encoding UDP-Galf-transporters. Deletion and localization studies indicate that UgtA and UgtB have redundant functions in the biosynthesis of Galf-containing glycoconjugates.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0541-2) contains supplementary material, which is available to authorized users.
Highlights
Galactofuranose (Galf)-containing glycoconjugates are present in numerous microbes, including filamentous fungi where they are important for morphology, virulence and maintaining cell wall integrity
Uridine diphosphate (UDP)-Galf transporters belong to the family of nucleotide sugar transporters (NSTs) and the genomes of aspergilli contain at least 12 subfamilies of NSTs [15]
Note that A. nidulans and A. fumigatus only possess a single UDP-Galf transporter, and deletion of this gene resulted in a Galf-negative phenotype [18, 19]
Summary
Galactofuranose (Galf)-containing glycoconjugates are present in numerous microbes, including filamentous fungi where they are important for morphology, virulence and maintaining cell wall integrity. The incorporation of Galf-residues into galactomannan, galactomannoproteins and glycolipids is carried out by Golgi-localized Galf transferases. The cell wall is an important extracellular structure of fungal species. It is essential for growth and survival as it protects the cell from lysis by its internal turgor pressure. The cell wall of filamentous fungi is composed of several different carbohydrate polymers (chitin, β-1,3-glucan, β-1,3/1,4-glucan, α-glucan, galactosaminogalactan and galactomannan) and glycoproteins (galactomannoproteins) [2]. The galactose in galactomannan and galactomannoproteins in filamentous fungi is present in the form of galactofuranose (Galf), the fivemembered ring form of this hexose. Because the presence of Galf is often associated with virulence in pathogenic bacteria, fungi and protozoan, it is considered as an important virulence factor and its biosynthesis as a target for antibiotics [4,5,6,7]
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