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Quantifying the importance of galactofuranose in Aspergillus nidulans hyphal wall surface organization by atomic force microscopy.

ABSTRACT: The fungal wall mediates cell-environment interactions. Galactofuranose (Galf), the five-member ring form of galactose, has a relatively low abundance in Aspergillus walls yet is important for fungal growth and fitness. Aspergillus nidulans strains deleted for Galf biosynthesis enzymes UgeA (UDP-glucose-4-epimerase) and UgmA (UDP-galactopyranose mutase) lacked immunolocalizable Galf, had growth and sporulation defects, and had abnormal wall architecture. We used atomic force microscopy and force spectroscopy to image and quantify cell wall viscoelasticity and surface adhesion of ugeA? and ugmA? strains. We compared the results for ugeA? and ugmA? strains with the results for a wild-type strain (AAE1) and the ugeB deletion strain, which has wild-type growth and sporulation. Our results suggest that UgeA and UgmA are important for cell wall surface subunit organization and wall viscoelasticity. The ugeA? and ugmA? strains had significantly larger surface subunits and lower cell wall viscoelastic moduli than those of AAE1 or ugeB? hyphae. Double deletion strains (ugeA? ugeB? and ugeA? ugmA?) had more-disorganized surface subunits than single deletion strains. Changes in wall surface structure correlated with changes in its viscoelastic modulus for both fixed and living hyphae. Wild-type walls had the largest viscoelastic modulus, while the walls of the double deletion strains had the smallest. The ugmA? strain and particularly the ugeA? ugmA? double deletion strain were more adhesive to hydrophilic surfaces than the wild type, consistent with changes in wall viscoelasticity and surface organization. We propose that Galf is necessary for full maturation of A. nidulans walls during hyphal extension.

SUBMITTER: Paul BC

PROVIDER: S-EPMC3127658 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Quantifying the importance of galactofuranose in Aspergillus nidulans hyphal wall surface organization by atomic force microscopy.

Paul Biplab C BC El-Ganiny Amira M AM Abbas Mariam M Kaminskyj Susan G W SG Dahms Tanya E S TE

Eukaryotic cell 20110218 5

The fungal wall mediates cell-environment interactions. Galactofuranose (Galf), the five-member ring form of galactose, has a relatively low abundance in Aspergillus walls yet is important for fungal growth and fitness. Aspergillus nidulans strains deleted for Galf biosynthesis enzymes UgeA (UDP-glucose-4-epimerase) and UgmA (UDP-galactopyranose mutase) lacked immunolocalizable Galf, had growth and sporulation defects, and had abnormal wall architecture. We used atomic force microscopy and force ...[more]

PMID: 21335527

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Project description:?-1,3-Glucan is one of the main polysaccharides in the cell wall of filamentous fungi. Aspergillus nidulans has two ?-1,3-glucan synthase genes, agsA and agsB. We previously revealed that AgsB is a major ?-1,3-glucan synthase in vegetative hyphae, but the function of AgsA remained unknown because of its low expression level and lack of phenotypic alteration upon gene disruption. To clarify the role of ?-1,3-glucan in hyphal aggregation, we constructed strains overexpressing agsA (agsAOE ) or agsB (agsBOE ), in which the other ?-1,3-glucan synthase gene was disrupted. In liquid culture, the wild-type and agsBOE strains formed tightly aggregated hyphal pellets, whereas agsAOE hyphae aggregated weakly. We analyzed the chemical properties of cell wall ?-1,3-glucan from the agsAOE and agsBOE strains. The peak molecular mass of ?-1,3-glucan from the agsAOE strain (1,480 ± 80 kDa) was much larger than that from the wild type (147 ± 52 kDa) and agsBOE (372 ± 47 kDa); however, the peak molecular mass of repeating subunits in ?-1,3-glucan was almost the same (after Smith degradation: agsAOE , 41.6 ± 5.8 kDa; agsBOE , 38.3 ± 3.0 kDa). We also analyzed localization of ?-1,3-glucan in the cell wall of the two strains by fluorescent labeling with ?-1,3-glucan-binding domain-fused GFP (AGBD-GFP). ?-1,3-Glucan of the agsBOE cells was clearly located in the outermost layer, whereas weak labeling was detected in the agsAOE cells. However, the agsAOE cells treated with ?-1,3-glucanase were clearly labeled with AGBD-GFP. These observations suggest that ?-1,3-glucan covered most of ?-1,3-glucan synthesized by AgsA, although a small amount of ?-1,3-glucan was still present in the outer layer. We also constructed a strain with disruption of the amyG gene, which encodes an intracellular ?-amylase that synthesizes ?-1,4-glucooligosaccharide as a primer for ?-1,3-glucan biosynthesis. In this strain, the hyphal pellets and peak molecular mass of ?-1,3-glucan (94.5 ± 1.4 kDa) were smaller than in the wild-type strain, and ?-1,3-glucan was still labeled with AGBD-GFP in the outermost layer. Overall, these results suggest that hyphal pellet formation depends on the molecular mass and spatial localization of ?-1,3-glucan as well as the amount of ?-1,3-glucan in the cell wall of A. nidulans.

Dataset Information

Quantifying the importance of galactofuranose in Aspergillus nidulans hyphal wall surface organization by atomic force microscopy.

Publications

Quantifying the importance of galactofuranose in Aspergillus nidulans hyphal wall surface organization by atomic force microscopy.

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