Aspergillus nidulans cell wall composition and function change in response to hosting several Aspergillus fumigatus UDP-galactopyranose mutase activity mutants

Abstract

Deletion or repression of Aspergillus nidulans ugmA (AnugmA), involved in galactofuranose biosynthesis, impairs growth and increases sensitivity to Caspofungin, a β-1,3-glucan synthesis antagonist. The A. fumigatus UgmA (AfUgmA) crystal structure has been determined. From that study, AfUgmA mutants with altered enzyme activity were transformed into AnugmA▵ to assess their effect on growth and wall composition in A. nidulans. The complemented (AnugmA::wild type AfugmA) strain had wild type phenotype, indicating these genes had functional homology. Consistent with in vitro studies, AfUgmA residues R182 and R327 were important for its function in vivo, with even conservative amino (RK) substitutions producing AnugmA? phenotype strains. Similarly, the conserved AfUgmA loop III histidine (H63) was important for Galf generation: the H63N strain had a partially rescued phenotype compared to AnugmA▵. Collectively, A. nidulans strains that hosted mutated AfUgmA constructs with low enzyme activity showed increased hyphal surface adhesion as assessed by binding fluorescent latex beads. Consistent with previous qPCR results, immunofluorescence and ELISA indicated that AnugmA▵ and AfugmA-mutated A. nidulans strains had increased α-glucan and decreased β-glucan in their cell walls compared to wild type and AfugmA-complemented strains. Like the AnugmA▵ strain, A. nidulans strains containing mutated AfugmA showed increased sensitivity to antifungal drugs, particularly Caspofungin. Reduced β-glucan content was correlated with increased Caspofungin sensitivity. Aspergillus nidulans wall Galf, α-glucan, and β-glucan content was correlated in A. nidulans hyphal walls, suggesting dynamic coordination between cell wall synthesis and cell wall integrity.

Figure 1. Colony morphology of Aspergillus nidulans wild type (WT) strain complemented with wild type AfUgmA (WC), single residue AfUgmA mutants (F66A, H63N, R182K, R182A, R327K, R327A) and AnugmAΔ strains, grown on complete medium at 30°C for 3 d.

The colour difference between WT and WC strains was due to slightly different ages of culture. See Figure S1 in File S1 for a direct comparison of the spore colours of these two strains.

Figure 2. Surface adhesion of Aspergillus nidulans strains to fluorescent latex beads.

Wild type (WT), strain complemented with wild type AfUgmA (WC), single residue AfUgmA mutants (F66A, H63N and R327A) and AnugmAΔ strains. Bar  =  20 µm is for all images.

Figure 3. In vivo distribution of GFP-tagged AfUgmA in Aspergillus nidulans.

A. Wild type complemented (WC) and single residue mutants (H63N, R182A and R327A) have comparable AfUgmA-GFP distribution. The single residue mutants have the ugmAΔ hyphal morphology. Bar  =  20 µm for all images. B. Confirmation of AfUgmA-GFP fusion protein distribution by Western blot. Total protein was extracted from A. nidulans wild type (WT; AAE1) and GFP-tagged AfUgmA strains (R182A-GFP, R327A-GFP, H63N-GFP, AnUgmA::AfUgm-GFP). Total Proteins (15 µg/lane) were separated on 10% SDS-PAGE and immunoblotted with an anti-GFP antibody.

Figure 4. Localization of Galactofuranose (Galf) in Aspergillus nidulans Cell wall.

A. Galf immunolocalization. B. Immunofluorescence quantification of Galf using confocal system software (see Methods). Error bar shows standard error. Aspergillus nidulans wild type (WT), wild type AfUgmA-complemented (WC), mutated AfUgmA (as listed), and AnugmAΔ. Bar  =  10 μm for all images.

Figure 5. Localization of Alpha-glucan in Aspergillus nidulans Cell wall.

A. Alpha-glucan immunolocalization. B. Immunofluorescence quantification of Alpha-glucan using confocal system software (see Methods). Error bar shows standard error. Aspergillus nidulans wild type (WT), wild type AfUgmA-complemented (WC), mutated AfUgmA (as listed), and AnugmAΔ. Bar  =  10 μm for all images.

Figure 6. Localization of Beta-glucan in Aspergillus nidulans Cell wall.

A. Beta-glucan immunolocalization. B. Immunofluorescence quantification of Beta-glucan using confocal system software (see Methods). Error bar shows standard error. Aspergillus nidulans wild type (WT), wild type AfUgmA-complemented (WC), mutated AfUgmA (as listed), and AnugmAΔ. Bar  =  10 μm for all images.