<i>In vivo</i> distribution of GFP-tagged <i>Af</i>UgmA in <i>Aspergillus nidulans</i>.

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

<i>Aspergillus nidulans</i> Cell Wall Composition and Function Change in Response to Hosting Several <i>Aspergillus fumigatus</i> UDP-Galactopyranose Mutase Activity Mutants

<div><p>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.</p></div

Localization of Beta-glucan in <i>Aspergillus nidulans</i> Cell wall.

<p>A. Beta-glucan immunolocalization. B. Immunofluorescence quantification of Beta-glucan using confocal system software (see Methods). Error bar shows standard error. <i>Aspergillus nidulans</i> wild type (WT), wild type <i>Af</i>UgmA-complemented (WC), mutated <i>Af</i>UgmA (as listed), and An<i>ugmAΔ</i>. Bar  =  10 μm for all images.</p

Structure-based sequence alignment of <i>A. nidulans</i>, <i>E. coli</i>, and Human forms of GALE.

<p>Identical residues are highlighted in red, similar residues are red surrounded with blue boxes.</p

<i>Aspergillus nidulans</i> wild type and chimaera cell wall carbohydrates^a, antifungal drug sensitivity^ac, and relative UGM activity^b.

<p>^a Absorbance at 405 nm was recorded using ELISA reader, Results are presented as an index of OD values with respect to wild type. Cell walls were isolated from <i>A. nidulans</i> strains after 48 h growth. Index sensitivity values are for each drug/strain combination compared to wild type. Index values > 1.0 were more sensitive than wild type. Index values that differed by > 0.2 were based on data that were significantly different.</p><p>^b Relative UGM activity <i>in vitro</i> with respect to wild type, derived from data in (33) and this study for F66A. nd, not determined; na, not available (low protein expression); bd, below detection.</p><p>^c Caspofungin, Casp; Itraconazole, Itra; Calcofluor White, CFW. See Materials and Methods for drug dosage and medium formulation. Drug sensitivity was measured using a disc diffusion assay as described in methods (Figure S3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085735#pone.0085735.s001" target="_blank">File S1</a>). For Caspofungin, sensitivity was the radius (mm) of the clear zone with no visible growth. Mean ± SE of two measurements for each of four biological replicates were used for statistical analysis (not shown).</p

Colony morphology of <i>Aspergillus nidulans</i> wild type (WT) strain complemented with wild type <i>Af</i>UgmA (WC), single residue <i>Af</i>UgmA mutants (F66A, H63N, R182K, R182A, R327K, R327A) and An<i>ugmA</i>Δ strains, grown on complete medium at 30°C for 3 d.

<p>The colour difference between WT and WC strains was due to slightly different ages of culture. See Figure S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085735#pone.0085735.s001" target="_blank">File S1</a> for a direct comparison of the spore colours of these two strains.</p

Proposed biosynthetic pathway of UDP-galactofuranose (UDP-Gal<i>f</i>) in <i>Aspergillus nidulans</i>.

<p>Proposed biosynthetic pathway of UDP-galactofuranose (UDP-Gal<i>f</i>) in <i>Aspergillus nidulans</i>.</p

Localization of Alpha-glucan in <i>Aspergillus nidulans</i> Cell wall.

<p>A. Alpha-glucan immunolocalization. B. Immunofluorescence quantification of Alpha-glucan using confocal system software (see Methods). Error bar shows standard error. <i>Aspergillus nidulans</i> wild type (WT), wild type <i>Af</i>UgmA-complemented (WC), mutated <i>Af</i>UgmA (as listed), and An<i>ugmAΔ</i>. Bar  =  10 μm for all images.</p

Schematic of the initial HPLC studies for AnGALE.

<p>a) Examination of UDP-Glc<i>p</i>/UDP-Gal<i>p</i> substrate interconversion for wild-type and mutant enzymes. b) UNGM coupled reaction for monitoring interconversion of the UDP-Glc<i>p</i>NAc/UDP-Gal<i>p</i>NAc substrate pair. Colors represent both product formation and the enzyme responsible for the catalytic reaction.</p

Interactions anchoring the cofactor and substrate within the active site of AnGALE.

<p>(<b>a</b>) <b>Schematic and (b) model representations of the UDP-Glc<i>p</i> binding</b>. (<b>c</b>) <b>Schematic and (d) model representation of the NAD⁺ binding</b>. For the schematic representations hydrogen bonds are shown in green and hydrophobic interactions in red. Hydrogen bonds are depicted as dashed lines in the model representations.</p