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

Microbial glycoside hydrolases as antibiofilm agents with cross-kingdom activity.

Galactosaminogalactan and Pel are cationic heteropolysaccharides produced by the opportunistic pathogens Aspergillus fumigatus and Pseudomonas aeruginosa, respectively. These exopolysaccharides both contain 1,4-linked N-acetyl-d-galactosamine and play an important role in biofilm formation by these organisms. Proteins containing glycoside hydrolase domains have recently been identified within the biosynthetic pathway of each exopolysaccharide. Recombinant hydrolase domains from these proteins (Sph3h from A. fumigatus and PelAh from P. aeruginosa) were found to degrade their respective polysaccharides in vitro. We therefore hypothesized that these glycoside hydrolases could exhibit antibiofilm activity and, further, given the chemical similarity between galactosaminogalactan and Pel, that they might display cross-species activity. Treatment of A. fumigatus with Sph3h disrupted A. fumigatus biofilms with an EC50 of 0.4 nM. PelAh treatment also disrupted preformed A. fumigatus biofilms with EC50 values similar to those obtained for Sph3h In contrast, Sph3h was unable to disrupt P. aeruginosa Pel-based biofilms, despite being able to bind to the exopolysaccharide. Treatment of A. fumigatus hyphae with either Sph3h or PelAh significantly enhanced the activity of the antifungals posaconazole, amphotericin B, and caspofungin, likely through increasing antifungal penetration of hyphae. Both enzymes were noncytotoxic and protected A549 pulmonary epithelial cells from A. fumigatus-induced cell damage for up to 24 h. Intratracheal administration of Sph3h was well tolerated and reduced pulmonary fungal burden in a neutropenic mouse model of invasive aspergillosis. These findings suggest that glycoside hydrolases can exhibit activity against diverse microorganisms and may be useful as therapeutic agents by degrading biofilms and attenuating virulence

Galectin-3 enhances neutrophil motility and extravasation into the airways during Aspergillus fumigatus infection.

Aspergillus fumigatus is an opportunistic mold that infects patients who are immunocompromised or have chronic lung disease, causing significant morbidity and mortality in these populations. While the factors governing the host response to A. fumigatus remain poorly defined, neutrophil recruitment to the site of infection is critical to clear the fungus. Galectin-3 is a mammalian β-galactose-binding lectin with both antimicrobial and immunomodulatory activities, however the role of galectin-3 in the defense against molds has not been studied. Here we show that galectin-3 expression is markedly up-regulated in mice and humans with pulmonary aspergillosis. Galectin-3 deficient mice displayed increased fungal burden and higher mortality during pulmonary infection. In contrast to previous reports with pathogenic yeast, galectin-3 exhibited no antifungal activity against A. fumigatus in vitro. Galectin-3 deficient mice exhibited fewer neutrophils in their airways during infection, despite normal numbers of total lung neutrophils. Intravital imaging studies confirmed that galectin-3 was required for normal neutrophil migration to the airspaces during fungal infection. Adoptive transfer experiments demonstrated that stromal rather than neutrophil-intrinsic galectin-3 was necessary for normal neutrophil entry into the airspaces. Live cell imaging studies revealed that extracellular galectin-3 directly increases neutrophil motility. Taken together, these data demonstrate that extracellular galectin-3 facilitates recruitment of neutrophils to the site of A. fumigatus infection, and reveals a novel role for galectin-3 in host defense against fungal infections

Inhibition or disruption of NETs attenuates the susceptibility of <i>A</i>. <i>nidulans</i> to killing by human neutrophil-mediated killing.

<p>(A) Neutrophil extracellular traps formation by primary human PMN as visualized by the DNA intercalating agent propidium iodide. Arrows indicate the increased binding of propidium iodide stained NETs on the surface of wild-type <i>A</i>. <i>nidulans</i> hyphae. Images were acquired using a 543 nm laser and detected by confocal microscopy at 600X magnification with 4X digital zoom. Scale bar represents 10 μm. (B) Susceptibility of fungal strains to injury by PMNs in the presence (gray bars) or absence (black bars) of micrococcal nuclease (MNase). (C) Susceptibility of fungal strains to injury by PMNs from healthy donor (black bars) or from a CGD patient (grey bars). (D) Susceptibility of fungal strains to injury by PMNs pre-treated with 10 μM dexamethasone (grey bars) or untreated (black bars). * indicates a significant difference between <i>A</i>. <i>nidulans</i> and <i>A</i>. <i>fumigatus</i> or An-Uge3 strains, p<0.05 by ANOVA with Tukey’s test for pairwise comparison. § indicates significant difference treatment groups of PMNs co-incubated with <i>A</i>. <i>nidulans</i>, p<0.05 by ANOVA with Tukey’s test for pairwise comparison. All data are represented as mean +/- SEM.</p

Overexpression of <i>uge3</i> or <i>ugeB</i> in <i>A</i>. <i>nidulans</i> increases the GalNAc content of GAG and enhances the formation of adherent biofilms.

<p>(A) Relative expression of <i>uge3</i> in the An-Uge3 strain and <i>ugeB</i> in the An-UgeB strain compared to the expression level of <i>ugeB</i> in wild-type <i>A</i>. <i>nidulans</i> grown in Brian medium and as measured by real-time RT-PCR. (B) Total amount of secreted GAG from the indicated strains. (C) GalNAc content of secreted GAG from the indicated strains as determined by gas chromatography and quantified by hexose or hexosamine assays. (D) Cell wall GalNAc staining with FITC-conjugated soybean agglutinin (SBA). SBA binding to mature hyphal mats of the indicated strains was quantified by fluorometry. (E) Scanning electron micrograph of hyphae of indicated species at 20,000X magnification. Arrows indicate surface decorations associated with cell wall-bound GAG. (F) Formation of adherent biofilms on tissue culture treated polystyrene plates by the indicated strains. After 24 hours growth, biofilms were washed and visualized by staining with 0.1% crystal violet. (G) Detection of β-1,3-glucan exposure on the surface of hyphae by immunostaining with Fc-dectin-1 antibody labeled with FITC secondary antibody and quantified by fluorometry at 495 nm. For all panels: An-Uge3 indicates the <i>A</i>. <i>nidulans</i> overexpressing <i>uge3</i> strain; An-UgeB indicates the <i>A</i>. <i>nidulans</i> overexpressing <i>ugeB</i> strain; and AnWT indicates wild type <i>A</i>. <i>nidulans</i>. Data are represented as mean +/- SEM and * indicates a significant difference between <i>A</i>. <i>nidulans</i>, and both overexpression strains, p<0.05 by ANOVA with Tukey’s test for pairwise comparison.</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

GAG-mediated resistance to neutrophil killing is dependent on neutrophil lysate content.

<p>Susceptibility of fungal strains to injury by lysates derived from, (A) primary human neutrophils, (B) primary human neutrophils treated with DPI., (C) primary C57BL/6 mouse neutrophils or <i>gp91</i>^<i>phox</i> deficient (CGD) mouse neutrophils. * indicates a significant difference between <i>A</i>. <i>nidulans</i> and <i>A</i>. <i>fumigatus</i> or An-Uge3 strains, p<0.05 by ANOVA with Tukey’s test for pairwise comparison.</p

<i>nidulans</i> produces GalNAc-poor GAG which is associated with non-adherence.

<p><b><i>A</i>.</b> (A) Galactose and GalNAc content of secreted GAG from either <i>A</i>. <i>fumigatus</i> or <i>A</i>. <i>nidulans</i> as identified by gas chromatography and quantified by hexose or hexosamine assays. (B) Formation of adherent biofilms on tissue culture-treated polystyrene plates by <i>A</i>. <i>fumigatus</i> and <i>A</i>. <i>nidulans</i>. After 24 hours growth, biofilms were washed and visualized by staining with 0.1% crystal violet. (C) Detection of β-1,3-glucan exposure on the surface of hyphae by immunostaining with Fc-dectin-1 antibody by fluorometry. (D) Relative expression of <i>ugeB</i> in <i>A</i>. <i>nidulans</i> and <i>uge3</i> in <i>A</i>. <i>fumigatus</i>, during growth in Brian medium as measured by real-time RT-PCR. Expression of <i>tef1</i> from each respective species was used as an internal reference gene. Primer efficiency was verified, and was not different between species (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005187#ppat.1005187.s002" target="_blank">S2E Fig</a>). For all panels: data are represented as mean +/- SEM. AfWT indicates <i>A</i>. <i>fumigatus</i>, and AnWT indicates <i>A</i>. <i>nidulans</i>. * indicates a significant difference between <i>A</i>. <i>fumigatus</i> and <i>A</i>. <i>nidulans</i>, p<0.05 by Student <i>t</i> test or ANOVA with Tukey’s test for pairwise comparison, where applicable.</p

GAG mediated enhancement of <i>A</i>. <i>nidulans</i> virulence requires functional leukocyte NADPH oxidase.

<p>(A) Survival of leukopenic mice infected with either <i>A</i>. <i>nidulans</i> or An-Uge3 conidia. N = 9 per infection group. (B) Survival of <i>gp91</i>^<i>phox</i> deficient mice lacking functional NADPH oxidase infected with either <i>A</i>. <i>nidulans</i> or An-Uge3 conidia. N = 17 per infection group. For all panels: An-Uge3 indicates the <i>A</i>. <i>nidulans</i> overexpressing <i>uge3</i> strain; AnWT indicates wild type <i>A</i>. <i>nidulans</i>; and AfWT indicates wild type <i>A</i>. <i>fumigatus</i>. n.s. indicates no significant difference in survival of <i>A</i>. <i>nidulans</i> compared with the An-Uge3, strain as determined by the Mantel-Cox log-rank test with pairwise comparison applying Bonferroni correction.</p