Brendan D. Snarr

Aspergillus Galactosaminogalactan Mediates Adherence to Host Constituents and Conceals Hyphal β-Glucan from the Immune System

Aspergillus fumigatus is the most common cause of invasive mold disease in humans. The mechanisms underlying the adherence of this mold to host cells and macromolecules have remained elusive. Using mutants with different adhesive properties and comparative transcriptomics, we discovered that the gene uge3, encoding a fungal epimerase, is required for adherence through mediating the synthesis of galactosaminogalactan. Galactosaminogalactan functions as the dominant adhesin of A. fumigatus and mediates adherence to plastic, fibronectin, and epithelial cells. In addition, galactosaminogalactan suppresses host inflammatory responses in vitro and in vivo, in part through masking cell wall β-glucans from recognition by dectin-1. Finally, galactosaminogalactan is essential for full virulence in two murine models of invasive aspergillosis. Collectively these data establish a role for galactosaminogalactan as a pivotal bifunctional virulence factor in the pathogenesis of invasive aspergillosis.

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.

Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms

Therapeutic enzyme treatment disrupts Pseudomonas biofilms, potentiating antibiotics and ameliorating the innate immune system. Bacterial biofilms present a significant medical challenge because they are recalcitrant to current therapeutic regimes. A key component of biofilm formation in the opportunistic human pathogen Pseudomonas aeruginosa is the biosynthesis of the exopolysaccharides Pel and Psl, which are involved in the formation and maintenance of the structural biofilm scaffold and protection against antimicrobials and host defenses. Given that the glycoside hydrolases PelAh and PslGh encoded in the pel and psl biosynthetic operons, respectively, are utilized for in vivo exopolysaccharide processing, we reasoned that these would provide specificity to target P. aeruginosa biofilms. Evaluating these enzymes as potential therapeutics, we demonstrate that these glycoside hydrolases selectively target and degrade the exopolysaccharide component of the biofilm matrix. PelAh and PslGh inhibit biofilm formation over a 24-hour period with a half maximal effective concentration (EC50) of 69.3 ± 1.2 and 4.1 ± 1.1 nM, respectively, and are capable of disrupting preexisting biofilms in 1 hour with EC50 of 35.7 ± 1.1 and 12.9 ± 1.1 nM, respectively. This treatment was effective against clinical and environmental P. aeruginosa isolates and reduced biofilm biomass by 58 to 94%. These noncytotoxic enzymes potentiated antibiotics because the addition of either enzyme to a sublethal concentration of colistin reduced viable bacterial counts by 2.5 orders of magnitude when used either prophylactically or on established 24-hour biofilms. In addition, PelAh was able to increase neutrophil killing by ~50%. This work illustrates the feasibility and benefits of using bacterial exopolysaccharide biosynthetic glycoside hydrolases to develop novel antibiofilm therapeutics.

Sph3 Is a Glycoside Hydrolase Required for the Biosynthesis of Galactosaminogalactan in Aspergillus fumigatus.

Background: The pathways governing biosynthesis of the Aspergillus fumigatus exopolysaccharide galactosaminogalactan are poorly understood. Results: The structure of Sph3 revealed a (β/α)8 barrel fold. The enzyme hydrolyzes galactosaminogalactan and is required for the synthesis of this exopolysaccharide. Conclusion: Sph3 is a glycoside hydrolase (GH) whose activity is essential for galactosaminogalactan biosynthesis. Significance: Sph3 defines a new glycoside hydrolase superfamily, GH family 135. Aspergillus fumigatus is the most virulent species within the Aspergillus genus and causes invasive infections with high mortality rates. The exopolysaccharide galactosaminogalactan (GAG) contributes to the virulence of A. fumigatus. A co-regulated five-gene cluster has been identified and proposed to encode the proteins required for GAG biosynthesis. One of these genes, sph3, is predicted to encode a protein belonging to the spherulin 4 family, a protein family with no known function. Construction of an sph3-deficient mutant demonstrated that the gene is necessary for GAG production. To determine the role of Sph3 in GAG biosynthesis, we determined the structure of Aspergillus clavatus Sph3 to 1.25 Å. The structure revealed a (β/α)8 fold, with similarities to glycoside hydrolase families 18, 27, and 84. Recombinant Sph3 displayed hydrolytic activity against both purified and cell wall-associated GAG. Structural and sequence alignments identified three conserved acidic residues, Asp-166, Glu-167, and Glu-222, that are located within the putative active site groove. In vitro and in vivo mutagenesis analysis demonstrated that all three residues are important for activity. Variants of Asp-166 yielded the greatest decrease in activity suggesting a role in catalysis. This work shows that Sph3 is a glycoside hydrolase essential for GAG production and defines a new glycoside hydrolase family, GH135.

Deacetylation of Fungal Exopolysaccharide Mediates Adhesion and Biofilm Formation.

ABSTRACT The mold Aspergillus fumigatus causes invasive infection in immunocompromised patients. Recently, galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetylgalactosamine (GalNAc), was identified as a virulence factor required for biofilm formation. The molecular mechanisms underlying GAG biosynthesis and GAG-mediated biofilm formation were unknown. We identified a cluster of five coregulated genes that were dysregulated in GAG-deficient mutants and whose gene products share functional similarity with proteins that mediate the synthesis of the bacterial biofilm exopolysaccharide poly-(β1-6)-N-acetyl-d-glucosamine (PNAG). Bioinformatic analyses suggested that the GAG cluster gene agd3 encodes a protein containing a deacetylase domain. Because deacetylation of N-acetylglucosamine residues is critical for the function of PNAG, we investigated the role of GAG deacetylation in fungal biofilm formation. Agd3 was found to mediate deacetylation of GalNAc residues within GAG and render the polysaccharide polycationic. As with PNAG, deacetylation is required for the adherence of GAG to hyphae and for biofilm formation. Growth of the Δagd3 mutant in the presence of culture supernatants of the GAG-deficient Δuge3 mutant rescued the biofilm defect of the Δagd3 mutant and restored the adhesive properties of GAG, suggesting that deacetylation is an extracellular process. The GAG biosynthetic gene cluster is present in the genomes of members of the Pezizomycotina subphylum of the Ascomycota including a number of plant-pathogenic fungi and a single basidiomycete species, Trichosporon asahii, likely a result of recent horizontal gene transfer. The current study demonstrates that the production of cationic, deacetylated exopolysaccharides is a strategy used by both fungi and bacteria for biofilm formation. IMPORTANCE This study sheds light on the biosynthetic pathways governing the synthesis of galactosaminogalactan (GAG), which plays a key role in A. fumigatus virulence and biofilm formation. We find that bacteria and fungi use similar strategies to synthesize adhesive biofilm exopolysaccharides. The presence of orthologs of the GAG biosynthetic gene clusters in multiple fungi suggests that this exopolysaccharide may also be important in the virulence of other fungal pathogens. Further, these studies establish a molecular mechanism of adhesion in which GAG interacts via charge-charge interactions to bind to both fungal hyphae and other substrates. Finally, the importance of deacetylation in the synthesis of functional GAG and the extracellular localization of this process suggest that inhibition of deacetylation may be an attractive target for the development of novel antifungal therapies. This study sheds light on the biosynthetic pathways governing the synthesis of galactosaminogalactan (GAG), which plays a key role in A. fumigatus virulence and biofilm formation. We find that bacteria and fungi use similar strategies to synthesize adhesive biofilm exopolysaccharides. The presence of orthologs of the GAG biosynthetic gene clusters in multiple fungi suggests that this exopolysaccharide may also be important in the virulence of other fungal pathogens. Further, these studies establish a molecular mechanism of adhesion in which GAG interacts via charge-charge interactions to bind to both fungal hyphae and other substrates. Finally, the importance of deacetylation in the synthesis of functional GAG and the extracellular localization of this process suggest that inhibition of deacetylation may be an attractive target for the development of novel antifungal therapies.

Divergent Targets of Aspergillus fumigatus AcuK and AcuM Transcription Factors during Growth In Vitro versus Invasive Disease

ABSTRACT In Aspergillus nidulans, the AcuK and AcuM transcription factors form a complex that regulates gluconeogenesis. In Aspergillus fumigatus, AcuM governs gluconeogenesis and iron acquisition in vitro and virulence in immunosuppressed mice. However, the function of AcuK was previously unknown. Through in vitro studies, we found that A. fumigatus ΔacuK single and ΔacuK ΔacuM double mutants had impaired gluconeogenesis and iron acquisition, similar to the ΔacuM mutant. Also, the ΔacuK, ΔacuM, and ΔacuK ΔacuM mutants had similar virulence defects in mice. However, the ΔacuK mutant had a milder defect in extracellular siderophore activity and induction of epithelial cell damage in vitro than did the ΔacuM mutant. Moreover, overexpression of acuM in the ΔacuK mutant altered expression of 3 genes and partially restored growth under iron-limited conditions, suggesting that AcuM can govern some genes independently of AcuK. Although the ΔacuK and ΔacuM mutants had very similar transcriptional profiles in vitro, their transcriptional profiles during murine pulmonary infection differed both from their in vitro profiles and from each other. While AcuK and AcuM governed the expression of only a few iron-responsive genes in vivo, they influenced the expression of other virulence-related genes, such as hexA and dvrA. Therefore, in A. fumigatus, while AcuK and AcuM likely function as part of the same complex, they can also function independently of each other. Furthermore, AcuK and AcuM have different target genes in vivo than in vitro, suggesting that in vivo infection stimulates unique transcriptional regulatory pathways in A. fumigatus.

Posaconazole-Loaded Leukocytes as a Novel Treatment Strategy Targeting Invasive Pulmonary Aspergillosis.

Background Impaired delivery of antifungals to hyphae within necrotic lesions is thought to contribute to therapeutic failure in invasive pulmonary aspergillosis (IPA). We hypothesized that transfusion of leukocytes loaded ex vivo with the lipophilic antifungal posaconazole could improve delivery of antifungals to the sites of established infection and improve outcome in experimental IPA. Methods The HL-60 leukemia cell line was differentiated to a neutrophil-like phenotype (differentiated HL-60 [dHL-60] cells) and then exposed to a range of posaconazole concentrations. The functional capacity and antifungal activity of these cells were assessed in vitro and in a mouse model of IPA. Results Posaconazole levels in dHL-60 cells were 265-fold greater than the exposure concentration. Posaconazole-loaded cells were viable and maintained their capacity to undergo active chemotaxis. Contact-dependent transfer of posaconazole from dHL-60 cells to hyphae was observed in vitro, resulting in decreased fungal viability. In a neutropenic mouse model of IPA, treatment with posaconazole-loaded dHL-60 cells resulted in significantly reduced fungal burden in comparison to treatment with dHL-60 cells alone. Conclusions Posaconazole accumulates at high concentrations in dHL-60 cells and increases their antifungal activity in vitro and in vivo. These findings suggest that posaconazole-loading of leukocytes may hold promise for the therapy of IPA.

Evolution of the Immune Response to Chronic Airway Colonization with Aspergillus fumigatus Hyphae

ABSTRACT Airway colonization by the mold Aspergillus fumigatus is common in patients with underlying lung disease and is associated with chronic airway inflammation. Studies probing the inflammatory response to colonization with A. fumigatus hyphae have been hampered by the lack of a model of chronic colonization in immunocompetent mice. By infecting mice intratracheally with conidia embedded in agar beads (Af beads), we have established an in vivo model to study the natural history of airway colonization with live A. fumigatus hyphae. Histopathological examination and galactomannan assay of lung homogenates demonstrated that hyphae exited beads and persisted in the lungs of mice up to 28 days postinfection without invasive disease. Fungal lesions within the airways were surrounded by a robust neutrophilic inflammatory reaction and peribronchial infiltration of lymphocytes. Whole-lung cytokine analysis from Af bead-infected mice revealed an increase in proinflammatory cytokines and chemokines early in infection. Evidence of a Th2 type response was observed only early in the course of colonization, including increased levels of interleukin-4 (IL-4), elevated IgE levels in serum, and a mild increase in airway responsiveness. Pulmonary T cell subset analysis during infection mirrored these results with an initial transient increase in IL-4-producing CD4+ T cells, followed by a rise in IL-17 and Foxp3+ cells by day 14. These results provide the first report of the evolution of the immune response to A. fumigatus hyphal colonization.

Microbial glycoside hydrolases as antibiofilm agents with cross-kingdom activity

Significance The production of biofilms is an important strategy used by both bacteria and fungi to colonize surfaces and to enhance resistance to killing by immune cells and antimicrobial agents. We demonstrate that glycoside hydrolases derived from the opportunistic fungus Aspergillus fumigatus and Gram-negative bacterium Pseudomonas aeruginosa can be exploited to disrupt preformed fungal biofilms and reduce virulence. Additionally, these glycoside hydrolases can be used to potentiate antifungal drugs by increasing their hyphal penetration, to protect human cells from fungal-induced injury, and attenuate virulence of A. fumigatus in a mouse model of invasive aspergillosis. The findings of this study identify recombinant microbial glycoside hydrolases as promising therapeutics with the potential for antibiofilm activity against pathogens across different taxonomic kingdoms. 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.

Immune Recognition of Fungal Polysaccharides

The incidence of fungal infections has dramatically increased in recent years, in large part due to increased use of immunosuppressive medications, as well as aggressive medical and surgical interventions that compromise natural skin and mucosal barriers. There are relatively few currently licensed antifungal drugs, and rising resistance to these agents has led to interest in the development of novel preventative and therapeutic strategies targeting these devastating infections. One approach to combat fungal infections is to augment the host immune response towards these organisms. The polysaccharide-rich cell wall is the initial point of contact between fungi and the host immune system, and therefore, represents an important target for immunotherapeutic approaches. This review highlights the advances made in our understanding of the mechanisms by which the immune system recognizes and interacts with exopolysaccharides produced by four of the most common fungal pathogens: Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, and Histoplasma capsulatum. Work to date suggests that inner cell wall polysaccharides that play an important structural role are the most conserved across diverse members of the fungal kingdom, and elicit the strongest innate immune responses. The immune system senses these carbohydrates through receptors, such as lectins and complement proteins. In contrast, a greater diversity of polysaccharides is found within the outer cell walls of pathogenic fungi. These glycans play an important role in immune evasion, and can even induce anti-inflammatory host responses. Further study of the complex interactions between the host immune system and the fungal polysaccharides will be necessary to develop more effective therapeutic strategies, as well as to explore the use of immunosuppressive polysaccharides as therapeutic agents to modulate inflammation.