The Fungal Exopolysaccharide Galactosaminogalactan Mediates Virulence by Enhancing Resistance to Neutrophil Extracellular Traps

Of the over 250 Aspergillus species, Aspergillus fumigatus accounts for up to 80% of invasive human infections. A. fumigatus produces galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetyl-galactosamine (GalNAc) that mediates adherence and is required for full virulence. Less pathogenic Aspergillus species were found to produce GAG with a lower GalNAc content than A. fumigatus and expressed minimal amounts of cell wall-bound GAG. Increasing the GalNAc content of GAG of the minimally pathogenic A. nidulans, either through overexpression of the A. nidulans epimerase UgeB or by heterologous expression of the A. fumigatus epimerase Uge3 increased the amount of cell wall bound GAG, augmented adherence in vitro and enhanced virulence in corticosteroid-treated mice to levels similar to A. fumigatus. The enhanced virulence of the overexpression strain of A. nidulans was associated with increased resistance to NADPH oxidase-dependent neutrophil extracellular traps (NETs) in vitro, and was not observed in neutropenic mice or mice deficient in NADPH-oxidase that are unable to form NETs. Collectively, these data suggest that cell wall-bound GAG enhances virulence through mediating resistance to NETs

Overlapping and Distinct Roles of Aspergillus fumigatus UDP-glucose 4-Epimerases in Galactose Metabolism and the Synthesis of Galactose-containing Cell Wall Polysaccharides* * This work was supported, in whole or in part, by National Institutes of Health Grant R01AI073829. This work was also supported by operating funds from the Canadian Institutes of Health Research.

The cell wall of Aspergillus fumigatus contains two galactose-containing polysaccharides, galactomannan and galactosaminogalactan, whose biosynthetic pathways are not well understood. The A. fumigatus genome contains three genes encoding putative UDP-glucose 4-epimerases, uge3, uge4, and uge5. We undertook this study to elucidate the function of these epimerases. We found that uge4 is minimally expressed and is not required for the synthesis of galactose-containing exopolysaccharides or galactose metabolism. Uge5 is the dominant UDP-glucose 4-epimerase in A. fumigatus and is essential for normal growth in galactose-based medium. Uge5 is required for synthesis of the galactofuranose (Galf) component of galactomannan and contributes galactose to the synthesis of galactosaminogalactan. Uge3 can mediate production of both UDP-galactose and UDP-N-acetylgalactosamine (GalNAc) and is required for the production of galactosaminogalactan but not galactomannan. In the absence of Uge5, Uge3 activity is sufficient for growth on galactose and the synthesis of galactosaminogalactan containing lower levels of galactose but not the synthesis of Galf. A double deletion of uge5 and uge3 blocked growth on galactose and synthesis of both Galf and galactosaminogalactan. This study is the first survey of glucose epimerases in A. fumigatus and contributes to our understanding of the role of these enzymes in metabolism and cell wall synthesis

Overlapping and distinct roles of Aspergillus fumigatus UDP-glucose 4-epimerases in galactose metabolism and the synthesis of galactose-containing cell wall polysaccharides.

The cell wall of Aspergillus fumigatus contains two galactose-containing polysaccharides, galactomannan and galactosaminogalactan, whose biosynthetic pathways are not well understood. The A. fumigatus genome contains three genes encoding putative UDP-glucose 4-epimerases, uge3, uge4, and uge5. We undertook this study to elucidate the function of these epimerases. We found that uge4 is minimally expressed and is not required for the synthesis of galactose-containing exopolysaccharides or galactose metabolism. Uge5 is the dominant UDP-glucose 4-epimerase in A. fumigatus and is essential for normal growth in galactose-based medium. Uge5 is required for synthesis of the galactofuranose (Galf) component of galactomannan and contributes galactose to the synthesis of galactosaminogalactan. Uge3 can mediate production of both UDP-galactose and UDP-N-acetylgalactosamine (GalNAc) and is required for the production of galactosaminogalactan but not galactomannan. In the absence of Uge5, Uge3 activity is sufficient for growth on galactose and the synthesis of galactosaminogalactan containing lower levels of galactose but not the synthesis of Galf. A double deletion of uge5 and uge3 blocked growth on galactose and synthesis of both Galf and galactosaminogalactan. This study is the first survey of glucose epimerases in A. fumigatus and contributes to our understanding of the role of these enzymes in metabolism and cell wall synthesis

Dectin-1 blockade abrogates the increased TNFα secretion induced by the GAG deficient Δ<i>uge3</i> mutant.

<p>BMDDCs were infected with hyphae of the indicated strains for 6 h, after which the TNFα content of culture supernatants was determined by EIA. Dectin-1 recognition of β-glucan was inhibited by preincubating BMDDCs with a monoclonal anti-dectin 1 antibody or by preincubating hyphae with Fc-dectin-1. Results are mean ± SEM of duplicate experiments, each performed in triplicate. * indicates a significant reduction of TNFα production compared to BMDDCs exposed to the Δ<i>uge3</i> mutant without dectin-1 blocking, <i>p</i><0.05 by factor ANOVA.</p

Uge3 is required for full virulence in a highly immunosuppressed mouse model of IA.

<p>(A) Survival of highly immunosuppressed mice treated with cyclophosphamide and cortisone acetate and infected with the indicated <i>A. fumigatus</i> strains. * indicates significantly increased survival of mice infected with the Δ<i>uge3</i> mutant as compared with those infected with the wild-type, <i>p</i> = 0.002 by the log rank test (<i>n</i> = 12 mice per fungal strain). (B) Quantification of fungal DNA in lung homogenates of mice after 5 days of infection. Results are median ± interquartile range of 8 mice per strain. * indicates a significantly reduced fungal DNA content in lungs of mice infected with the Δ<i>uge3</i> mutant as compared with those infected with the wild-type strain, <i>p</i> = 0.11 by the Wilcoxon rank sum test. (C) Photomicrographs of Gomori menthenamine silver stained sections of mouse lungs obtained 4 days after infection with the wild-type strain Af293. No fungal lesions could be identified in the lungs of mice infected with the Δ<i>uge3</i> mutant strain. Magnification was ×100 and ×400. White arrows indicate hyphae. Note the lack of infiltrating leukocytes within fungal lesions.</p

The <i>A. fumigatus</i> Δ<i>uge3</i> mutant induces a hyperinflammatory response in non-neutropenic mice that is attenuated in highly immunocompromised mice.

<p>(A.) Corticosteroid treated mice were infected by inhalation with the indicated strains of <i>A. fumigatus</i> and sacrificed three days after infection. Fungal burden was determined by pulmonary galactomannan content and pulmonary inflammation was measured by determining MPO, and TNF-α content. Pulmonary injury was quantified by measuring LDH release in BAL fluid. MPO, TNF-α, and LDH levels were normalized to the fungal burden of each strain in Panel 1. Results are median ± interquartile range of 8 mice per strain. * indicates a significant decrease in fungal burden or a significant increase in MPO, TNFα or LDH content in lungs of mice infected with the Δ<i>uge3</i> mutant as compared to the lungs of mice infected with the wild-type strain, <i>p</i><0.01 by the Wilcoxon rank sum test. (B) Corticosteroid and cyclophosphamide treated mice were infected by inhalation with the indicated strains, sacrificed and the lungs processed as in (A). MPO, TNF-α, and LDH levels were normalized to the fungal burden of each strain in Panel 1. Results are median ± interquartile range of 9 mice per strain. Note: y-axis values for all graphs are lower than those in (A).</p

Uge3 is required for full virulence in mice model of IA.

<p>(A) Survival of cortisone acetate-treated Balb/C mice infected with the indicated <i>A. fumigatus</i> strains. Data are the combined results of 2 independent experiments for a total of 16 mice per strain. * indicates a significant increase in survival between mice infected with the Δ<i>uge3</i> mutant and mice infected with the wild-type or with the <i>uge3</i> complemented strain, <i>p</i> = 0.017 and <0.001 respectively by the log rank test. (B) Quantification of fungal DNA in lung homogenates of mice after 4 days of infection. Results are median ± interquartile of 8 mice per strain. * indicates a significantly reduced fungal DNA content in lungs of mice infected with the Δ<i>uge3</i> mutant compared with those infected with the wild-type strain, <i>p</i> = 0.015 by the Wilcoxon rank sum test. (C) Photomicrographs of PAS stained sections of mouse lungs obtained 4 days after infection with the indicated strains. White arrows indicate hyphae, black arrows indicate infiltrating leukocytes.</p

Production of GalNAc-rich GAG correlates with reported virulence of <i>Aspergillus spp</i>.

<p>(A) Scanning electron micrograph of hyphae of indicated species at 20,000X magnification. Arrows indicate surface decorations associated with cell wall-bound GAG. The GAG deficient <i>A</i>. <i>fumigatus</i> Δ<i>uge3</i> mutant [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005187#ppat.1005187.ref010" target="_blank">10</a>] and the <i>A</i>. <i>fumigatus</i> Δ<i>stuA</i> mutant [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005187#ppat.1005187.ref049" target="_blank">49</a>] which produces only minimal amounts of GAG are included for comparison purposes. (B) Cell wall GalNAc staining with FITC-conjugated soybean agglutinin (SBA). SBA binding to mature hyphal mats of the indicated species was quantified by fluorometry. Data are represented as mean +/- SEM. * indicates a significant difference between <i>A</i>. <i>fumigatus</i> and other species, p<0.05 by ANOVA and pairwise comparison.</p

Dendritic cells produce an increased pro-inflammatory cytokine profile in response to the GAG deficient mutant.

<p>Graphs show the cytokine content of culture supernatant after 6 h of infection of BMDDCs with hyphae of the indicated strains. LPS was used as a positive control, and medium as a negative control. Cytokine concentrations in culture supernatants were determined by multiplex EIA. Results are mean ± standard error of duplicate determination of cytokine concentrations, indicated in pg/mL. * indicates a significantly increased cytokine concentration induced by the Δ<i>uge3</i> mutant, as compared with the one induced by Af293, <i>p</i><0.05 by factor ANOVA. § indicates that actual value is above 5,000 pg TNFα/mL (measures exceeded upper limit of the test).</p

Sensitivity of fungal strains to cell wall perturbing agents.

<p>Sensitivity of fungal strains to cell wall perturbing agents.</p