Functional and molecular characterization of Aspergillus fumigatus phosphoglucomutase (Pgm): a potential target for antifungal therapy

TL;DR

This study explores a key enzyme in the fungus Aspergillus fumigatus and shows it could be a target for new antifungal drugs.

Contribution

The study provides new functional and molecular insights into AfPGM and validates it as a potential antifungal drug target.

Findings

Reduced AfPGM activity impairs fungal growth and cell wall-related traits.

AfPGM complements a yeast mutant, confirming functional conservation.

The compound ISFP10 inhibits AfPGM and disrupts fungal phenotypes.

Abstract

Introduction.
Aspergillus fumigatus phosphoglucomutase (AfPGM) is a central enzyme in fungal carbohydrate metabolism that catalyses the interconversion of glucose-1-phosphate and glucose-6-phosphate, generating precursors such as uridine diphosphate glucose required for the synthesis of key cell wall polysaccharides.

Hypothesis/Gap Statement. Although AfPGM has been implicated in fungal viability and cell wall integrity, further functional and molecular characterization is required to better define its role in cell wall-associated processes and to evaluate its potential as a selective antifungal target.

Aim. This study aimed to further characterize the functional contribution of AfPGM to fungal growth, cell wall-related phenotypes, biofilm formation and enzymatic activity.

Methodology. A conditional A. fumigatus PalcA::pgm mutant was used to assess growth, soluble β-glucan levels and biofilm formation. Functional complementation was evaluated by heterologous expression of Afpgm in a Saccharomyces cerevisiae pgm2Δ strain. Enzymatic inhibition was examined using the isothiazolone compound ISFP10.

Results. Reduced AfPGM activity was associated with impaired growth, altered cell wall-related phenotypes, decreased soluble β-glucan levels and diminished biofilm biomass. Expression of Afpgm restored growth and sedimentation defects in the yeast pgm2Δ mutant to WT levels, confirming functional conservation. ISFP10 inhibited AfPGM activity and disrupted associated fungal phenotypes.

Conclusion. These findings further define the functional role of AfPGM in fungal carbohydrate metabolism and cell wall-associated biology. The selective inhibition of AfPGM relative to human phosphoglucomutase supports its potential as a promising target for the development of novel antifungal therapies.

Figures

In eukaryotic cells, PGM is vital to major metabolic pathways. Glc, glucose; Gal-1-P, galactose-1-phosphate; Glc-1-P, glucose-1-phosphate; Glc-6-P, glucose-6-phosphate. Figure adapted from [7].

Sequence alignment of A. fumigatus (AfPGM) and H. sapiens (HsPGM), performed using clustalw (MacVector, Inc. 18.6.1), demonstrates significant amino acid homology. Identical residues are shaded in dark grey, conserved residues in light grey and a black star indicates the fungal conserved cysteine C353 in AfPGM.

Evaluation of growth characteristics of PalcA::pgm mutant and KU80 WT strains of A. fumigatus, monitored using a 96-well microplate reader over 72 h at 37 °C in AMM liquid medium, supplemented with 100 mM threonine (AMMT) (a), and in 100 mM threonine plus 6 µM glucose (AMMTG) (b). Conidia concentrations for both strains ranged from 106 to 104 cells ml−1. Data are presented as means+sd from three independent experiments. Statistical significance: P<0.0001.

TEM of the ultrastructural effects of A. fumigatus PalcA::pgm mutant and KU80 WT conidia grown after 72 h of growth at 37 °C in AMM liquid supplemented with 100 mM threonine plus 6 µM glucose (AMMTG). The WT strain exhibited dense cytoplasm and a uniform cell wall (CW) coated with fibrillar structures (FS) (a and b), whereas the mutant strain showed less dense cytoplasm, irregular mitochondria and an absence of fibrillar structures on the cell wall periphery. Scale bars: 400 nm (40,000× magnification); 100 nm (100,000× magnification).

Functional complementation of Saccharomyces cerevisiae pgm2Δ by A. fumigatus Afpgm cDNA. WT S. cerevisiae or S. cerevisiae pgm2Δ were transformed with either the pYES2.1/lacZ control expression vector alone or pYES2.1 containing Afpgm cDNA, as indicated. Cells were grown after 18 h at 30 °C on synthetic solid medium containing 2% glucose (lacking uracil) or 2% galactose (-URA) as the sole carbon source. The numbers below the OD600nm represent spectrophotometer readings starting at 0.3 OD 600nm, followed by serial 10-fold dilutions of the yeast cultures.

Relative growth curves of WT S. cerevisiae and pgm2Δ strain transformed with either the pYES2.1 control expression vector or pYES2.1 containing A. fumigatus Afpgm cDNA, as indicated. Cell growth was initiated in synthetic -URA medium supplemented with 2% glucose and incubated for 18 h at 30 °C. After incubation, yeast cells were pelleted, resuspended in synthetic -URA medium containing 2% galactose and adjusted to an OD600nm of 0.4. Cells were then incubated for an additional 18 h at 30 °C in 2% galactose, and OD600nm was measured to assess growth (a). Sedimentation rates (OD600nm) were subsequently measured over an 8-h time period in 2 % galactose (b). Error bars represent the mean±sd of three independent experiments. One-way ANOVA: P<0.01.

PGM activity in A. fumigatus PalcA::pgm mutant and KU80 WT strains. Cells were grown in AMM liquid medium supplemented with either 100 mM threonine (AMMT) or 100 mM threonine plus 6 µM glucose (AMMTG) at 37 °C for 7 days with shaking at 250 r.p.m. PGM activity (measured as nM glucose-6-phosphate) in protein homogenates was determined as described in the ‘Methods’ section. Data are expressed as means+sem of three independent experiments: P<0.01.

Effect of PGM on soluble β-glucan levels in A. fumigatus PalcA::pgm mutant and KU80 WT strains. Cells were grown in AMM liquid medium supplemented with either 100 mM threonine (AMMT) or 100 mM threonine plus 6 µM glucose (AMMTG) at 37 °C for 7 days with shaking at 250 r.p.m. Soluble β-glucan content (pg ml−1) in protein homogenates was quantified as described in the ‘Methods’ section. Data are expressed as means±sem from three independent experiments. P<0.05.

Biofilm formation of A. fumigatus PalcA::pgm mutant and KU80 WT strains. Cells (105 to 104 cells ml−1) were incubated in a polystyrene 96-well plate for 48 h at 37 °C to assess adhesion ability. Unbound cells were removed; wells were washed three times with 1× PBS and stained with 0.5% crystal violet plus 2.5% glutaraldehyde. Wells were then repeatedly washed with 1× PBS and photographed (a). Biofilm formation was quantified by measuring crystal violet staining at OD600nm (b). Data are expressed as means+sem from three independent experiments: *P<0.05, P<0.0001.

Dose-dependent inhibition of A. fumigatus PGM activity by ISFP10 in yeast extracts. Yeast extracts were prepared from the S. cerevisiae pgm2Δ strain transformed with Afpgm cDNA. AfPGM activity was measured in the presence of increasing concentrations of ISFP10 (6.25–200 µM). Data represent means+sem of four independent experiments: P<0.01, *P<0.001, P<0.0001.

Full text

Introduction

Invasive aspergillosis (IA) infections, particularly due to Aspergillus fumigatus, remain a serious threat to immunocompromised and immunosuppressed patients despite current antifungal therapies, with mortality rates ranging from 30 to 95 % [13]. Given the limited options of available drugs and the rise of antifungal resistance [4], developing novel antifungal targets and therapeutic strategies against this fungal pathogen is critically important. A potential approach lies in exploiting essential fungal pathways that are absent or significantly different in human hosts, thereby minimizing host toxicity [5]. One such critical pathway is carbohydrate metabolism, fundamental to fungal growth, cell wall integrity and virulence [68].

Phosphoglucomutase (PGM) is an essential enzyme in A. fumigatus metabolism of carbohydrates, catalysing the reversible interconversion of glucose-1-phosphate (G1P) and glucose-6-phosphate (G6P). This reaction is significant because G1P is a precursor to the synthesis of uridine diphosphate glucose (UDP-glucose), necessary for carbohydrate and glycoconjugate generation. Additionally, G1P and uridine triphosphate, a nucleotide glucose activation energy source [9], are converted into UDP-glucose and pyrophosphate by UDP-glucose pyrophosphoglucomutase, as shown in modified Fig. 1 [7]. This pathway is a vital component for the synthesis of various fungal polysaccharides, including β-glucans and galactomannan, which are major constituents of the fungal cell wall [1011]. In A. fumigatus, the hyphae cell wall contains polysaccharides including chitin, glucan, galactomannan and galactosaminogalactan (GAG), all of which play important roles in the pathogenesis of invasive aspergillosis [1113]. β-1,3-Glucan constitutes a significant part of the carbohydrate composition in the cell wall of A. fumigatus [14] and is important for growth and cell wall integrity [1516]. Although PGM enzyme activity is believed to play a critical role in the formation of A. fumigatus biofilms due to its involvement in β-1,3-glucan biosynthesis [1719], definitive genetic evidence supporting this role remains lacking. The biofilm extracellular matrix confers enhanced resistance to antifungal agents and host immune defenses, contributing significantly to persistent and difficult-to-treat infections [2021]. Furthermore, biofilm formation involves the production of polysaccharides whose synthesis depends on the availability of specific sugar precursors generated through PGM activity [2223]. Recently, the discovery of the small molecule isothiazolone inhibitor ISFP10, which selectively inhibits A. fumigatus PGM with a 50-fold preference over the human homologue (HsPGM), represents a new promising approach for antifungal therapy [19]. Such inhibitors have the potential to disrupt cell wall biosynthesis and biofilm formation, offering a novel therapeutic strategy to combat this pathogen and improve clinical outcomes for patients with fungal infections.

In eukaryotic cells, PGM is vital to major metabolic pathways. Glc, glucose; Gal-1-P, galactose-1-phosphate; Glc-1-P, glucose-1-phosphate; Glc-6-P, glucose-6-phosphate. Figure adapted from [7].

In this study, we characterized the functional and molecular properties of the A. fumigatus PGM to validate its potential as a target for antifungal intervention. First, consistent with previous studies, we observe a significant growth defect of the A. fumigatus pgm-deficient mutant (P_alcA_::pgm) compared to the WT (KU80) strain, which we quantify under defined culture conditions, using the same threonine-inducing and glucose-repressing conditions published by these authors [1]. Furthermore, consistent with the same report [1], TEM analysis showed an apparent increase in cell wall thickness and altered cell wall architecture in the PalcA::pgm mutant compared to the WT strain, supporting a role for PGM in cell wall organization and structure. In contrast to the confirmatory phenotypic analyses described above, the following experiments represent new contributions to the field. Using a heterologous expression system in Saccharomyces cerevisiae pgm2Δ cells [2] complemented with Afpgm under the GAL promoter, we demonstrated that Afpgm restores growth in both liquid and solid media in the presence of galactose. Using this same system, we further showed that expression of Afpgm in the S. cerevisiae pgm2Δ strain fully rescued the sedimentation defect to levels comparable with the WT parental strain. In addition, we show for the first time that the P_alcA_::pgm mutant exhibits significantly reduced PGM enzymatic activity relative to the WT strain. Quantitative analysis of β-glucan content further revealed a significant reduction in this key cell wall carbohydrate in the P_alcA_::pgm mutant compared to the WT strain. Moreover, disruption of Afpgm significantly impaired biofilm extracellular matrix production in the P_alcA_::pgm mutant, a phenotype not previously reported. Finally, we demonstrate that the established fungal PGM inhibitor ISFP10 significantly reduces A. fumigatus PGM activity in a dose-dependent manner using the heterologous yeast expression system described above.

Collectively, these newly described functional, biochemical and pharmacological analyses establish AfPGM as a promising and selective target for antifungal drug development, an especially important advance considering the increasing threat of antifungal resistance.

Methods

In vitro analysis, fungal strains and growth media

The PGM amino acid sequences from A. fumigatus (XP_754438.1) (AfPGM) and Homo sapiens (AAA60080.1) (HsPGM) were aligned using the clustalw algorithm within MacVector version 18.6.1 software (MacVector, Inc., Cary, NC). WT KU80 and knock-out (KO) P_alcA_::pgm strains of A. fumigatus were obtained from the Centre for Gene Regulation and Expression, University of Dundee, UK. Transformed S. cerevisiae WT (BY4742) or S. cerevisiae pgm2Δ (YMR105C) with the pYES2.1 TOPO TA expression vector [7] and the expression control plasmid pYES2.1/V5-His/lacZ, and S. cerevisiae pgm2Δ restored with the A. fumigatus Afpgm were used for yeast complementation and sedimentation assays. A. fumigatus strains KU80 and P_alcA_::pgm were cultured on solid Aspergillus minimal media (AMM) (Formedium, Norfolk, UK). All S. cerevisiae cultures were grown in yeast extract–pepton–dextrose minimal medium containing 2 % glucose or 2 % galactose lacking uracil (URA-).

Growth assay of Ku80 and PalcA::pgm

A. fumigatus strains were grown on solid AMM at 37 °C for 7 days. Conidia were harvested with 1× PBS containing 0.005 % Tween 20 and filtered through a 40-µm cell strainer to remove mycelium and hyphae. Conidia (1×10^6^ cells ml^−1^) were counted using a Burker counting chamber [24]. The respective spores were pelleted and resuspended in separate liquid AMM, supplemented with 100 mM threonine (AMMT) and 100 mM threonine with 6 mM glucose (AMMTG) (19) as carbon sources. Spores were diluted from 10^6^ to 10^4^ cells ml^−1^, and 200 µl was dispensed into a 96-well plate in triplicate, respectively. The optical density at 600 nm (OD600_nm_) was measured in a microplate reader spectrophotometer (Molecular Devices, CA, USA) at 37 °C for 72 h. Three independent experimental runs were performed, and the relative growth rate was evaluated as the percentage of OD600_nm_ at the concentration relative to the mean OD600_nm_ in the absence of inoculum.

TEM analysis of A. fumigatus KU80 and PalcA::pgm cell wall

Fresh WT and KO strains were grown in liquid AMMTG for 72 h at 37 °C with shaking at 250 r.p.m. TEM sample preparation was performed in parallel for both strains as described previously with minor modifications [3]. Cultures from three independent biological experiments were processed separately. For each experiment, multiple ultrathin sections were prepared, and ~25–30 individual cells per strain were examined by transmission electron microscopy. Representative images are shown. Cells were pelleted by centrifugation at 8,000 r.p.m. for 5 min and fixed using 4% paraformaldehyde plus 2 % glutaraldehyde in 0.1 M phosphate buffer (pH 7.0) for 24 h at 4 °C. Samples were encapsulated in 1% low-melting agar and processed through 0.15 M cacodylate buffer, 2% osmium tetroxide, 2% aqueous uranyl acetate, graded ethanol dehydration and Spurr resin infiltration. Ultrathin sections (100 nm) were obtained, stained with lead citrate, rinsed with sterile distilled water and examined using an HT7800-120 kV transmission electron microscope (Hitachi HighTech, IL, USA).

No morphometric quantification was performed; observations were qualitative and focused on comparative structural features between strains.

Functional complementation of A. fumigatus pgm in S. cerevisiae pgm2Δ

The full-length cDNA for Afpgm was amplified using a synthetic full-length Afpgm template (Integrated DNA Technologies). Amplification was performed using Platinum Taq Polymerase High Fidelity (Invitrogen) and the following PCR primers: Afpgm Forward-5′-ATG TCG GTT CAA ACT GTT TCC AT-3′ and Afpgm Reverse-5′-TTA GGT TTT AAC ATC AGG GTC TTC-3′. The PCR product was ligated into the pYES2.1 TOPO TA yeast expression vector (Invitrogen), sequenced and transformed into E. coli One Shot^™^ TOP10F’ chemically competent cells. The resulting expression plasmid was then subcloned into a pgm2Δ-deficient yeast strain (YMR105C). Both this mutant strain and the parental BY4742 strain were also transformed with the control vector pYES2.1/V5-his/lacZ, following the manufacturer’s instructions [7]. For complementation analysis, transformants were grown overnight in liquid minimal medium lacking uracil and supplemented with 2% glucose or galactose. Cultures were serially diluted 1:10 (starting from OD600_nm_=0.3) and plated on synthetic agar medium lacking uracil, with 2% galactose or glucose.

Growth curves in S. cerevisiae

The yeast strains were grown in 5 ml of 2% glucose minimal medium lacking uracil at 30 °C for 18 h (220 r.p.m.), reaching mid-logarithmic phase as determined by OD600 measurements [2]. Cultures were adjusted to an OD600 of 0.4 to standardize the starting inoculum, pelleted at 2500 r.p.m. for 5 min and resuspended in 10 ml of 2% galactose minimal medium lacking uracil. Cultures were then incubated at 30 °C for 18 h (220 r.p.m.). OD600 measurements were blank-corrected against media-only controls. Growth was calculated relative to the standardized starting OD_600_ (0.4), representative cumulative growth over the 18 h induction period. Values were averaged from three independent biological replicates. Statistical analysis was performed as described in the ‘Statistical analyses’ section.

Sedimentation assay of A. fumigatus pgm in S. cerevisiae

The yeast strains listed above were grown in 10 ml of 2% galactose minimal media without uracil at 30 °C (220 r.p.m.) for 18 h. Cultures of each strain were diluted to an OD600_nm_ of 1.0 with fresh 2% minimal media and galactose in a 1 ml cuvette. The OD600_nm_ was measured every hour for 8 h using undisturbed cuvettes with the SpectraMax M2 spectrophotometer (Molecular Devices, CA, USA).

Analysis of Ku80 and PalcA::pgm PGM enzyme activity

Fresh A. fumigatus Ku80 and P_alcA_::pgm strains were separately grown in liquid AMMTG at 37 °C for 7 days, at 250 r.p.m. Cultures were filtered through a 40-µm cell strainer and used for protein lysate isolation. Equal wet weights of 100 mg mycelia were resuspended in 2-ml microcentrifuge tubes containing 300 µl of a mixed reagent of Yeast-Protein Extraction Reagent and Complete Protease Inhibitor Cocktail Tablets (Roche Diagnostics, IN, USA) (Thermo Fisher, Waltham, MA). The samples were disrupted with the Branson 450 Digital Sonifier (3×30 s each) (Marshall Scientific, NH, USA). Immediately, protein lysates were quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher, Inc.) and protein concentrations measured in a microplate reader at OD562_nm_. Protein aliquots of three separate individual experiments were stored at −20 °C until PGM activity analysis. The PGM enzymatic activities of Ku80 and P_alcA_::pgm grown in AMMTG were assessed as previously described [7]. For each strain, 1.0 µg protein was assayed with a phosphoglucomutase activity assay kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions.

β-Glucan analysis of A. fumigatus KU80 and PalcA::pgm

Freeze-dried mycelial biomass (100 mg wet weight) from KU80 and P_alcA_::pgm strains grown in AMMTG was resuspended in 300 µl of Y-PER™ Yeast Protein Extraction Reagent supplemented with Protease Inhibitor Cocktail Tablets (Roche Diagnostics, IN). Although Y-PER is commonly used for yeast, efficient protein recovery from A. fumigatus was achieved through combined chemical lysis and mechanical disruption. Samples were subjected to probe sonication using a Branson 450 Digital Sonifier (Marshall Scientific, NH) to ensure complete cell wall disruption. Lysates were clarified by centrifugation, and supernatants were collected for downstream enzymatic assays. Protein extraction efficiency and reproducibility were confirmed across independent biological replicates.

Protein concentration was performed on a microplate reader at OD562_nm_ using the Pierce BCA Protein Assay Kit. Protein aliquots of three repeated experimental runs were stored at −20 °C until use. The β-glucan content of the two strains was determined using the Endpoint Assay of the Glucatell^®^ Kit (Associates of Cape Cod, Inc., MA). Briefly, 50 µl of different glucan standard concentrations (5–40 pg ml^−1^) and 50 µl of each protein substrate (100 µg) were pipetted in duplicate into a 96-well microtiter plate. To each well, 50 µl of Glucatell reagent was added, and the plate was mixed by gently tapping the edge of the cover lid. The plate was preheated to 37±1 °C for 1 h in a microplate reader, and the reaction was then stopped by sequentially adding 50 µl of sodium nitrite, ammonium sulfamate and N-(1-Napthyl) ethylenediamine dihydrochloride. Finally, the absorption of the resulting colour development was measured in a microplate reader at OD545_nm_. The endpoint standard curve generated from glucan standard concentrations was used to quantify the β-glucan content.

Biofilm formation in A. fumigatus KU80 and PalcA::pgm

Fresh cultured KU80 and P_alcA_::pgm conidia grown in AMMTG were used to determine the biofilm-forming capacity, comparing the strains [2526]. To accomplish this, 200 µl of conidia (10^6^–10^4^ cells ml^−1^) suspended in AMMTG was pipetted into a polystyrene 96-well plate in triplicate and incubated for 48 h at 37 °C. After biofilms formed, wells were gently decanted and washed twice with 1× PBS, and the wells were gently air dried. Biofilms were stained with 100 µl per well of 0.5% (wt/vol) crystal violet (Sigma-Aldrich, MO), containing 2.5 % (vol/vol) glutaraldehyde, and the 96-well plate was incubated at room temperature for 30 min. The unbound stain was washed twice with 1× PBS and air-dried. After drying, 200 µl of 95% methanol was added to each well to release the bound dye from the biofilm without shaking. Subsequently, the absorbance of each well was measured with a microplate reader at OD600_nm_ [26].

Effects of isothiazolone ISFP10 on Afpgm PGM activity in S. cerevisiae

S. cerevisiae pgm2Δ (YMR105C) transformed with pYES2.1 and Afpgm cDNA were grown in 5 ml of 2 % glucose minimal media lacking uracil at 30 °C with shaking at 220 r.p.m. for 24 h [7]. Yeast cells were diluted to an OD 1.0 (600_nm_) and were pelleted at 2,500 r.p.m. for 5 min. and resuspended in 10 ml of 2 % galactose minimal medium lacking uracil. Cultures were then incubated for 18 h at 30 °C with shaking at 220 r.p.m. After incubation, cells were pelleted again at 2,500 r.p.m. for 5 min. Protein lysates were prepared from each cell pellet as described above, and total protein concentrations were determined using the Pierce BCA protein assay kit (7). Aliquots were stored at −20 °C until use.

To determine the potential effects of ISFP10 on PGM activity, 2.0 µg of the yeast protein was incubated with ISFP10 at various concentrations (200, 100, 50, 25, 12.5 and 6.25 µM) and maintained in 3 % DMSO following previously described methods [7]. Absorbance at 450_nm_ was measured 5 min after the reaction was initiated.

Statistical analyses

Statistical analysis and graphing were performed using GraphPad Prism for Windows (version 10.0.0; GraphPad Software, Inc., MA, USA). Data are presented as mean±sd. Differences between groups were assessed by ANOVA, followed by multiple comparison tests. The levels of statistical significance are indicated as follows: *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.

Results

PGM homologues in A. fumigatus and H. sapiens

The S. cerevisiae S288C strain contains two genes, pgm1 (NP_012795.1) and pgm2 (NP_013823.1), which encode PGMs. Among these, pgm2 accounts for more than 90% of total PGM activity in the cell [2728]. Our blastp analysis comparing A. fumigatus AfPGM and S. cerevisiae S288C ScPGM2 revealed 62% sequence similarity. Previous studies have shown AfPGM is conserved, with an amino acid substitution relative to HsPGM [19], a pattern also observed in other fungal pathogens [78]. These findings are consistent with our protein alignment analysis of AfPGM and HsPGM (Fig. 2), which shows that the cysteine residue (C353, marked with a black star) is not conserved in human HsPGM.

Sequence alignment of A. fumigatus (AfPGM) and H. sapiens (HsPGM), performed using clustalw (MacVector, Inc. 18.6.1), demonstrates significant amino acid homology. Identical residues are shaded in dark grey, conserved residues in light grey and a black star indicates the fungal conserved cysteine C353 in AfPGM.

Role of PGM in A. fumigatus viability and cell wall integrity

We employed a P_alcA_::pgm (KO) strain compared with the WT KU80 strain to qualitatively determine the effects of growth of AfPGM on A. fumigatus growth. The strains were cultured in liquid AMMTG and AMMT at 37 °C for 48 h. Yan et al. [19] successfully constructed this conditional mutant by replacing the native pgm promoter with the alcohol dehydrogenase promoter (PalcA) from A. nidulans. The PalcA promoter is highly regulated, induced by threonine and repressed by glucose [2930]. Yan et al. [19] showed that the KU80 WT strain was induced in the presence of 100 mM threonine or 100 mM threonine and 6 mM glucose, while the P_alcA_::pgm strain was partially inhibited on solid minimal media supplemented with 100 mM threonine and 6 mM glucose, suggesting that PalcA repression can inhibit the conditional mutant strain.

In our study, the P_alcA_::pgm strain displayed growth comparable to the KU80 WT in liquid AMM supplemented with 100 mM threonine (Fig. 3a). However, when grown in AMM with 100 mM threonine and 6 mM glucose, the P_alcA_::pgm strain showed markedly reduced growth relative to the WT KU80 strain (Fig. 3b). These findings indicate that while both strains grow similarly in threonine alone, glucose significantly suppresses the growth of the P_alcA_::pgm strain, supporting the finding that repression of AfPGM has a substantial impact on fungal growth.

*Evaluation of growth characteristics of PalcA::pgm mutant and KU80 WT strains of A. fumigatus, monitored using a 96-well microplate reader over 72 h at 37 °C in AMM liquid medium, supplemented with 100 mM threonine (AMMT) (a), and in 100 mM threonine plus 6 µM glucose (AMMTG) (b). Conidia concentrations for both strains ranged from 106 to 104 cells ml−1. Data are presented as means+sd from three independent experiments. Statistical significance: ***P<0.0001.

AfPGM regulates cell wall architecture

Transmission electron analysis (TEM) analysis of the A. fumigatus P_alcA_::pgm strain was further compared to the WT KU80 strain grown in AMMTG to qualitatively assess the role of PGM in maintaining cell wall structure. Surprisingly, the TEM images revealed that the P_alcA_::pgm mutant lacking AfPGM had a cell wall approximately four times thicker than that of the WT KU80 strain (Fig. 4). The KU80 strain exhibited dense cytoplasm, an intact nucleus, normal mitochondria, well-organized organelles and a uniform cell wall coated with fibrillar structures (Fig. 4a, b). In contrast, the cytoplasm of the P_alcA_::pgm mutant displayed less dense cytoplasm, irregular or swollen mitochondria and a disrupted cell wall, with aberrant glycoprotein or fibrillar structures on the outer cell wall (Fig. 4c, d). These findings indicate that the loss of PGM activity resulted in substantial alterations to the cell wall architecture.

TEM of the ultrastructural effects of A. fumigatus PalcA::pgm mutant and KU80 WT conidia grown after 72 h of growth at 37 °C in AMM liquid supplemented with 100 mM threonine plus 6 µM glucose (AMMTG). The WT strain exhibited dense cytoplasm and a uniform cell wall (CW) coated with fibrillar structures (FS) (a and b), whereas the mutant strain showed less dense cytoplasm, irregular mitochondria and an absence of fibrillar structures on the cell wall periphery. Scale bars: 400 nm (40,000× magnification); 100 nm (100,000× magnification).

Previous studies have also shown that morphological changes in fungi can result from disruptions in associated cell signalling pathways, suggestive of the abnormalities observed here [3133]. A key phenotypic feature of the P_alcA_::pgm mutant was the apparent absence of the hydrophobin layer, which is critical for assembling the rodlet layer. Disruption of hydrophobins can lead to decreased fungal virulence by eliminating this immune-masking layer [34]. Therefore, our findings suggest that the P_alcA_::pgm mutant exhibits significant alterations in both cell wall structure and internal cellular architecture [19].

A. fumigatus pgm can functionally complement pgm2-deficient yeast growth

We confirmed that Afpgm encodes a functional phosphoglucomutase by assessing its ability to complement the S. cerevisiae pgm2Δ strain (Fig. 5). The S. cerevisiae WT, pgm2Δ strain carrying the pYES2.1 control expression vector and pgm2Δ expressing Afpgm cDNA all exhibited similar growth on 2% glucose solid media lacking uracil after 18 h at 30 °C. However, the pgm2Δ yeast strain carrying the control vector exhibited inhibited growth on galactose medium lacking uracil. In contrast, restored growth was observed in both the Afpgm-expressing strain and WT with the control vector, which displayed similar growth profiles on glucose media.

Functional complementation of Saccharomyces cerevisiae pgm2Δ by A. fumigatus Afpgm cDNA. WT S. cerevisiae or S. cerevisiae pgm2Δ were transformed with either the pYES2.1/lacZ control expression vector alone or pYES2.1 containing Afpgm cDNA, as indicated. Cells were grown after 18 h at 30 °C on synthetic solid medium containing 2% glucose (lacking uracil) or 2% galactose (-URA) as the sole carbon source. The numbers below the OD600nm represent spectrophotometer readings starting at 0.3 OD 600nm, followed by serial 10-fold dilutions of the yeast cultures.

Evaluation of the pgm2Δ yeast strain expressing Afpgm cDNA in 2% galactose liquid media at 30 °C for 18 h demonstrated fully restored growth to levels comparable with the WT yeast strain carrying the vector control. This finding supports the conclusion that Afpgm functions as a bona fide phosphoglucomutase enzyme necessary for growth and viability (Fig. 6a). In addition, measurement of sedimentation rates in yeast strains cultured in 2% galactose for 18 h revealed a significantly increased sedimentation rate in the pgm2Δ strain yeast with vector control over the 8 h period (Fig. 6b). Enhanced sedimentation rates generally reflect altered fungal cell wall structure and composition. Complementation of Scpgm2Δ with Afpgm restored the sedimentation rate to levels comparable to the WT strain, further supporting the role of Afpgm as a phosphoglucomutase important for A. fumigatus cell wall structure. We acknowledge that Afpgm was expressed from the GAL promoter using the pYES2.1 plasmid system, which may result in elevated expression relative to endogenous chromosomal levels, and thus, we cannot exclude potential contributions of plasmid copy number or inducible overexpression. However, both mutant and control strains were transformed with the same vector backbone and analysed under identical induction conditions. Importantly, Afpgm expression restored defective growth and sedimentation phenotypes to WT levels without evidence of enhanced growth beyond WT controls, supporting functional enzymatic complementation rather than a nonspecific overexpression artefact.

*Relative growth curves of WT S. cerevisiae and pgm2Δ strain transformed with either the pYES2.1 control expression vector or pYES2.1 containing A. fumigatus Afpgm cDNA, as indicated. Cell growth was initiated in synthetic -URA medium supplemented with 2% glucose and incubated for 18 h at 30 °C. After incubation, yeast cells were pelleted, resuspended in synthetic -URA medium containing 2% galactose and adjusted to an OD600nm of 0.4. Cells were then incubated for an additional 18 h at 30 °C in 2% galactose, and OD600nm was measured to assess growth (a). Sedimentation rates (OD600nm) were subsequently measured over an 8-h time period in 2 % galactose (b). Error bars represent the mean±sd of three independent experiments. One-way ANOVA: *P<0.01.

Impaired PGM enzyme activity in the A. fumigatus Afpgm KO strain

PGM enzymatic activity assays revealed a significant reduction in PGM activity in the P_alcA_::pgm mutant compared to the WT strain. Specifically, the mutant exhibited approximately a 10-fold lower PGM activity than the parent strain when measured using equal concentrations of protein lysates (Fig. 7). This reduction indicates that the PalcA promoter effectively controls pgm gene expression, resulting in a marked decrease in PGM synthesis [35]. Similar reductions in PGM activity have also been reported in other microbes, such as Fusarium oxysporum [36] and Salmonella enterica [36], highlighting the critical role of PGM in microbial metabolic function.

*PGM activity in A. fumigatus PalcA::pgm mutant and KU80 WT strains. Cells were grown in AMM liquid medium supplemented with either 100 mM threonine (AMMT) or 100 mM threonine plus 6 µM glucose (AMMTG) at 37 °C for 7 days with shaking at 250 r.p.m. PGM activity (measured as nM glucose-6-phosphate) in protein homogenates was determined as described in the ‘Methods’ section. Data are expressed as means+sem of three independent experiments: *P<0.01.

Effects of pgm inhibit β-glucan synthesis in A. fumigatus

Previous studies have demonstrated that the absence of a functional or mutated pgm gene in A. fumigatus impairs or abolishes PGM activity, resulting in reduced conversion of G6P to G1P and, consequently, a deficiency in UDP-glucose synthesis [193738]. We sought to determine whether this deficiency results in the inhibition of β-glucan biosynthesis. In this analysis, we hypothesized that the P_alcA_::pgm KO strain would exhibit altered β-glucan content.

Our results show that the P_alcA_::pgm KO strain exhibits a ~4.4-fold reduction in soluble β-glucan content relative to the WT strain (Fig. 8). This reduction suggests that impaired PGM activity affects β-glucan production or processing, consistent with the observed defects in cell wall architecture. These data support an important role for PGM in pathways contributing to fungal cell wall integrity.

Effect of PGM on soluble β-glucan levels in A. fumigatus PalcA::pgm mutant and KU80 WT strains. Cells were grown in AMM liquid medium supplemented with either 100 mM threonine (AMMT) or 100 mM threonine plus 6 µM glucose (AMMTG) at 37 °C for 7 days with shaking at 250 r.p.m. Soluble β-glucan content (pg ml−1) in protein homogenates was quantified as described in the ‘Methods’ section. Data are expressed as means±sem from three independent experiments. P<0.05.

Impact of Afpgm on biofilm formation in A. fumigatus

A. fumigatus biofilms contribute significantly to pathogenesis by protecting antifungal drugs and host immune defenses, particularly in immunocompromised patients [4]. In this study, biofilm formation was quantitatively assessed in the A. fumigatus WT KU80 strain and the P_alcA_::pgm mutant, revealing a significant impairment in the mutant’s ability to form mature biofilms (Fig. 9).

*Biofilm formation of A. fumigatus PalcA::pgm mutant and KU80 WT strains. Cells (105 to 104 cells ml−1) were incubated in a polystyrene 96-well plate for 48 h at 37 °C to assess adhesion ability. Unbound cells were removed; wells were washed three times with 1× PBS and stained with 0.5% crystal violet plus 2.5% glutaraldehyde. Wells were then repeatedly washed with 1× PBS and photographed (a). Biofilm formation was quantified by measuring crystal violet staining at OD600nm (b). Data are expressed as means+sem from three independent experiments: *P<0.05, ***P<0.0001.

Using a crystal violet staining assay to quantify total biofilm biomass [56], the P_alcA_::pgm mutant consistently exhibited a statistically significant reduction in biofilm formation compared to the robust biofilms produced by the WT strain under identical conditions. While this assay does not distinguish among individual extracellular matrix components, these findings suggest that PGM activity contributes to biofilm development through its broader role in fungal carbohydrate metabolism.

Recent studies have identified GAG, rather than β-glucan, as a dominant polysaccharide component of the A. fumigatus biofilm extracellular matrix [7]. Given that PGM functions upstream in central carbohydrate metabolism, disruption of Afpgm may affect the biosynthesis or availability of multiple polysaccharide precursors, including those required for GAG, mannose-containing glycans and other matrix constituents. Thus, the observed biofilm defect in the P_alcA::_pgm mutant likely reflects a global impairment of extracellular matrix production rather than a β-glucan-specific effect.

Inhibition of A. fumigatus PGM activity by isothiazolone ISFP10

PGM in A. fumigatus is an essential enzyme involved in carbohydrate metabolism and cell wall biosynthesis, making it an attractive target for novel antifungal therapies [71939]. Yan et al. identified an isothiazolone fragment that selectively binds to a cysteine residue unique to AfPGM and absent in the human PGM (HsPGM), leading to the development of ISFP10, a derivative compound that inhibits AfPGM with an IC_50_ of 2 µM and exhibits onefold greater potency against AfPGM than HsPGM.

In our study, we evaluated ISFP10 for its inhibitory effect on AfPGM activity using yeast extracts. A recent study successfully cloned and characterized the AfPGM enzyme from A. fumigatus (AfPGM) by generating a conditional, repressible Afpgm mutant, confirming that AfPGM activity is essential for fungal growth and cell wall integrity [19]. Building on these findings, we tested ISFP10 in yeast extracts derived from the pgm2Δ yeast strain transformed to express Afpgm. As shown in Fig. 10, our results demonstrate that ISFP10 significantly inhibits AfPGM enzyme activity in a dose-dependent manner across a concentration range of 200 to 6.25 µM. Lastly, our lab has recently reported in mice the preclinical and toxicology assessment of ISFP10 in mice. These reports revealed no inherent safety or toxicology concerns with the defined experimental methods [89], indicating the potential utility of targeting A. fumigatus PGM with ISFP10 as a therapeutic strategy for treating those with A. fumigatus infections.

*Dose-dependent inhibition of A. fumigatus PGM activity by ISFP10 in yeast extracts. Yeast extracts were prepared from the S. cerevisiae pgm2Δ strain transformed with Afpgm cDNA. AfPGM activity was measured in the presence of increasing concentrations of ISFP10 (6.25–200 µM). Data represent means+sem of four independent experiments: **P<0.01, ***P<0.001, ***P<0.0001.

Discussion

A. fumigatus has been recognized by the World Health Organization (WHO) as a critical fungal pathogen on its priority list [40]. This ubiquitous pathogen is prevalent in both developed and developing countries [44142], posing a significant threat to human health [2]. While advanced diagnostic tools, prophylaxis and treatment have moderately reduced the number of IA cases [4345], the incidence is increasing among immunocompetent patients undergoing immunosuppressive treatments [4647]. Furthermore, although current anti-aspergillosis treatments are relatively effective, mortality remains high, largely due to the emergence of antifungal drug resistance in A. fumigatus [44849]. For instance, mortality rates in haematological patients range from 50 to 60% [50] and reach 48% in non-Cornavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis cases [51].

In this study, building on previous work, we provide further evidence for and a characterization of AfPGM. Our findings underscore the critical role of AfPGM in the organism’s life cycle and cell wall structure, suggesting that AfPGM may serve as a promising novel therapeutic target. Moreover, these findings are consistent with previous studies conducted on A. fumigatus, Pneumocystis jirovecii and Pneumocystis murina [7], and other top fungal pathogens identified by the WHO [40], revealing that eight out of ten fungal species contained a conserved cysteine (C353) in their respective PGM molecules [19]. Notably, this cysteine is absent from the HsPGM, making it a potentially viable and selective target for a pan-fungal targeted approach.

Our work further highlights the critical role of PGM enzymes in fungal pathogens. These findings suggest that targeting fungal PGM proteins could represent a promising strategy for the development of novel antifungal agents. Specific inhibitors, such as ISFP10, may disrupt downstream synthesis of both glucans and N-linked mannoproteins, thereby offering a novel therapeutic approach against A. fumigatus [19] and other fungal pathogens. Supporting this, previous studies have shown that the PGM-deficient mutants of Sanghuangporus vaninii exhibit reduced hyphal growth, increased polysaccharide accumulation and a significant decrease in β-1,3-glucan levels compared to the WT strain [52].

Taken together, these results underscore the essential role of PGM in fungal carbohydrate metabolism and cell wall biosynthesis, further demonstrating its potential as a valuable target for the development of new antifungal therapies against A. fumigatus and other life-threatening fungal infections.