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.

Overlapping and Distinct Roles of Aspergillus fumigatus UDP-glucose 4-Epimerases in Galactose Metabolism and the Synthesis of Galactose-containing Cell Wall Polysaccharides

“…Similar silent gene duplications have been reported in other cell wall-related genes in A. fumigatus (64,65). Interestingly, silencing of epimerases in Aspergillus species may play a role in virulence because in the nonpathogenic species A. nidulans, ugeB, the ortholog of A. fumigatus uge3, has been reported to be silent (55).…”

“…11). Although the genome of A. nidulans contains ugeB, an ortholog of uge3, this gene has been reported to be silent (55) and as a result likely does not contribute to UDPgalactose/UDP-glucose interconversion in this species.…”

“…For example, deletion of ugm1 in A. fumigatus resulted not only in an absence of galactomannan but also an increase in galactosaminogalactan synthesis and biofilm adherence (6). Although galactosaminogalactan production has not been studied in A. nidulans, it was reported that the deletion of ugmA and ugeA was associated with an increase in adherence (55), suggesting a similar phenomenon may also occur in this organism. One possibility is that impaired galactomannan synthesis results in accumulation of precursors that are then redirected to the galactosaminogalactan pathway.…”

D-galactose catabolism inPenicillium chrysogenum: Expression analysis of the structural genes of the Leloir pathway

“…In A. fumigatus, two structurally related UDP-hexose 4-epimerases were recently shown to be required for the synthesis of the galactosaminogalactan exopolysaccharide content of the fungal cell wall [26]. The weakly expressed A. nidulans gene for the paralog enzyme, ugeB, had been identified in an earlier work [32]. We included its P. chrysogenum ortholog as well as three additional autoannotated epimerase genes structurally related to A. nidulans loci AN0746 and AN3119 [17] in our current study to assess their possible role in Leloir catabolism of D-galactose next to ugeA.…”

Viscoelasticity in Biological Systems: A Special Focus on Microbes

“…The Young's modulus of microbial cells is determined from the non-linear portion of the force indentation curve with equations 2 or 3 [41]. given distance, and is used to measure cantilever deflection and to calculate sample indentation [39].…”