| Summary: | β-(1→5)-Galactofuranosyl residues are widely distributed in the subphylum Pezizomycotina of the phylum Ascomycota. Pezizomycotina includes many plant and animal pathogens. Although the structure of β-(1→5)-galactofuranosyl residues of galactomannans in filamentous fungi was discovered long ago, it remains unclear which enzyme is responsible for biosynthesis of this glycan. Fungal cell wall formation processes are complicated, and information concerning glycosyltransferases is essential for understanding them. In this study, we showed that GfsA and GfsC are responsible for the biosynthesis of all β-(1→5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans. The data presented here indicate that β-(1→5)-galactofuranosyl residues are involved in cell growth, conidiation, polarity, and cell surface hydrophobicity. Our new understanding of β-(1→5)-galactofuranosyl residue biosynthesis provides important novel insights into the formation of the complex cell wall structure and the virulence of the members of the subphylum Pezizomycotina.The pathogenic fungus Aspergillus fumigatus contains galactomannans localized on the surface layer of its cell walls, which are involved in various biological processes. Galactomannans comprise α-(1→2)-/α-(1→6)-mannan and β-(1→5)-/β-(1→6)-galactofuranosyl chains. We previously revealed that GfsA is a β-galactofuranoside β-(1→5)-galactofuranosyltransferase involved in the biosynthesis of β-(1→5)-galactofuranosyl chains. In this study, we clarified the biosynthesis of β-(1→5)-galactofuranosyl chains in A. fumigatus. Two paralogs exist within A. fumigatus: GfsB and GfsC. We show that GfsB and GfsC, in addition to GfsA, are β-galactofuranoside β-(1→5)-galactofuranosyltransferases by biochemical and genetic analyses. GfsA, GfsB, and GfsC can synthesize β-(1→5)-galactofuranosyl oligomers at up to lengths of 7, 3, and 5 galactofuranoses within an established in vitro highly efficient assay of galactofuranosyltransferase activity. Structural analyses of galactomannans extracted from ΔgfsB, ΔgfsC, ΔgfsAC, and ΔgfsABC strains revealed that GfsA and GfsC synthesized all β-(1→5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans and that GfsB exhibited limited function in A. fumigatus. The loss of β-(1→5)-galactofuranosyl residues decreased the hyphal growth rate and conidium formation ability and increased the abnormal hyphal branching structure and cell surface hydrophobicity, but this loss is dispensable for sensitivity to antifungal agents and virulence toward immunocompromised mice.
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