Role of β-galactofuranose and β-glucan in Aspergillus nidulans hyphal cell wall ultrastructure and physical properties

Paul, Biplab Chandra
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Faculty of Graduate Studies and Research, University of Regina

The fungal cell wall is a first line of defence from the external environment or chemical treatments. Glucan, chitin and mannan are the main components of the Aspergillus nidulans hyphal cell wall. The sugar β-galactofuranose is a minor component of the cell wall and thought to be responsible for cross-linking of the other cell wall components, therefore responsible for maintaining cell wall structural integrity. We investigated the role of β-galactofuranose on the structure and physical properties of the hyphal cell wall. Based on its unique capacity to image live samples, atomic force microscopy was used to determine both the ultrastructure and physical properties of the hyphal cell wall. Five different knockout strains of Aspergillus nidulans (ugeAΔ, ugeBΔ, ugeAΔ,ugeBΔ and ugmAΔ, ugeAΔ,ugmAΔ and ugtAΔ) associated with β-galactofuranose synthesis were compared with the wildtype strain (AAE1). Atomic force microscopic imaging and force spectroscopy of the mutant and wild type strains suggest that a lack of galactofuranose reduces the integrity of cell wall components, where the surface subunits of ugeAΔ and ugmAΔ are two-four fold larger than that of the wildtype (AAE1) respectively. The ugeBΔ strain shows similar sized subunits as the AAE1 strain, in contrast with the double mutant (ugeAΔ,ugeBΔ) which exhibits a fibrous cell surface structure. The ugtAΔ mutant strain, able to synthesize β-Galf but unable to incorporate the sugar into the cell wall, showed a similar surface structure to the double mutant ugeAΔ,ugmAΔ, with the largest surface subunits. The structural changes of the cell wall surface are accompanied by a change in cell wall viscoelasticity, where the cell wall of the wild type strain is the most viscoelastic in comparison to that of mutant strains, and the lowest cell wall viscoelasticity can be attributed to the complete absence of β-Galf. Live and fixed cell walls of the ugmAΔ, ugtAΔ, ugeAΔ,ugeBΔ, and ugeAΔ,ugmAΔ strains had extremely low viscoelastic moduli, which we attributed to a limitation of the model used to accurately calculate viscoelasticity. Moreover, the impaired cell wall packing in mutant strains is consistent with greater surface hydrophilicity for mutant strains compared to wild type. We propose that the lack of galactofuranose disrupts the proper packing of cell wall components, giving rise to more disordered surface subunits and therefore greater deformability. Topographic images of glucanase- and laminarinase-treated wildtype strains suggest that glucan is at least one component of the cell surface subunits. Mutant strains which lack Galf were more susceptible to laminarinase treatment, which we attribute to deeper enzyme penetration into the more loosely packed cell walls.

A Thesis Submitted to the Faculty of Graduate Studies and Research In Partial Fulfillment of the Requirements for the Degree of Master of Science in Biochemistry, University of Regina. xi , 91 l.