Fundamentals of growth, storage, genetics and microscopy of Aspergillus nidulans

This is a compendium of protocols for using Aspergillus nidulans in genetic, molecular, and cell biological investigations, originally written for members of my research group. It also summarizes our common growth media and nutritional supplements, many of which originally appeared elsewhere but now are difficult to locate. All solutions, tools, etc. are assumed to be sterile. All water should be sterile distilled or equivalent. Temperatures are in °C. Strains are available from the Fungal Genetics Stock Center (http://www.fgsc.net/)

Elucidation of substrate specificity in Aspergillus nidulans UDP-galactose-4-epimerase.

The frequency of invasive fungal infections has rapidly increased in recent years. Current clinical treatments are experiencing decreased potency due to severe host toxicity and the emergence of fungal drug resistance. As such, new targets and their corresponding synthetic pathways need to be explored for drug development purposes. In this context, galactofuranose residues, which are employed in fungal cell wall construction, but are notably absent in animals, represent an appealing target. Herein we present the structural and biochemical characterization of UDP-galactose-4-epimerase from Aspergillus nidulans which produces the precursor UDP-galactopyranose required for galactofuranose synthesis. Examination of the structural model revealed both NAD(+) and UDP-glucopyranose were bound within the active site cleft in a near identical fashion to that found in the Human epimerase. Mutational studies on the conserved catalytic motif support a similar mechanism to that established for the Human counterpart is likely operational within the A. nidulans epimerase. While the K m and k cat for the enzyme were determined to be 0.11 mM and 12.8 s(-1), respectively, a single point mutation, namely L320C, activated the enzyme towards larger N-acetylated substrates. Docking studies designed to probe active site affinity corroborate the experimentally determined activity profiles and support the kinetic inhibition results

Characterization of mannitol in Curvularia protuberata hyphae by FTIR and Raman spectromicroscopy †

FTIR and Raman spectromicroscopy were used to characterize the composition of Curvularia protuberata hyphae, and to compare a strain isolated from plants inhabiting geothermal soils with a non-geothermal isolate. Thermal IR source images of hyphae have been acquired with a 64 Â 64 element focal plane array detector; single point IR spectra have been obtained with synchrotron source light. In some C. protuberata hyphae, we have discovered the spectral signature of crystalline mannitol, a fungal polyol with complex protective roles. With FTIR-FPA imaging, we have determined that the protein content in cells remains fairly constant throughout the length of a hypha, whereas the mannitol is found at discrete, irregular locations. This is the first direct observation of mannitol in intact fungal hyphae. Since the concentration of mannitol in cells varies with respect to position and is not present in all hyphae, this discovery may be related to habitat adaptation, fungal structure and growth stages

<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

<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

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

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