Discovery of N-Glycan Outer Chain like α-(1→6)-linked Mannan Structures in Aspergillus fumigatus Mycelium

The cellular surface of the pathogenic filamentous fungus Aspergillus fumigatus is enveloped in a mannose layer, featuring well-established fungal-type galactomannan and O-mannose type galactomannan. This study reports the discovery of cell wall component in the mycelium that resemble N-glycan outer chains found in yeast. The glycosyltransferases involved in their biosynthesis in A. fumigatus were identified, with a focus on two key α-(1→2)-mannosyltransferases, Mnn2 and Mnn5, and two crucial α-(1→6)-mannosyltransferases, Mnn9 and Van1. In vitro examination revealed the roles of Mnn2 and Mnn5 in transferring α-(1→2)-mannosyl residues. 1H-NMR analysis of cell wall extracts from the Δ mnn2 Δ mnn5 strain indicated the existence of an α-(1→6)-linked mannan backbone in the mycelium, with Mnn2 and Mnn5 adding α-(1→2)-mannosyl residues to this backbone. The α-(1→6)-linked mannan-derived chemical shift was absent in strains where mnn9 or van1 was disrupted in the parental Δ mnn2 Δ mnn5 strain. Co-expressed Mnn9 and Van1 functioned as α-(1→6)-linked mannan polymerases in heterodimers, suggesting their crucial role in biosynthesizing the α-(1→6)-linked mannan backbone. Disruptions in these mannosyltransferases did not affect fungal-type galactomannan biosynthesis. The study provides insights into the complexity of fungal cell wall architecture and a comprehensive understanding of mannan biosynthesis in A. fumigatus, including the presence of outer chain like mannan structures and their potential role in cell wall integrity. Importance This study unravels the complexities of mannan biosynthesis in A. fumigatus, a key area for antifungal drug discovery. It reveals the presence of outer chain like mannan structures, offering fresh insights into the fungal cell wall’s design. Key enzymes Mnn2, Mnn5, Mnn9, and Van1 are instrumental in this process, with Mnn2 and Mnn5 adding specific mannose residues and Mnn9 and Van1 assembling the outer chain like structures. While fungal-type galactomannan’spresence in the cell wall is known, the existence of outer chain like mannan adds a new dimension to our understanding. This intricate web of mannan biosynthesis opens avenues for further exploration and enhances our understanding of fungal cell wall dynamics, paving the way for targeted drug development.