Aspergillus fumigatus tryptophan metabolic route differently affects host immunity.

Indoleamine 2,3-dioxygenases (IDOs) degrade l-tryptophan to kynurenines and drive the de novo synthesis of nicotinamide adenine dinucleotide. Unsurprisingly, various invertebrates, vertebrates, and even fungi produce IDO. In mammals, IDO1 also serves as a homeostatic regulator, modulating immune response to infection via local tryptophan deprivation, active catabolite production, and non-enzymatic cell signaling. Whether fungal Idos have pleiotropic functions that impact on host-fungal physiology is unclear. Here, we show that Aspergillus fumigatus possesses three ido genes that are expressed under conditions of hypoxia or tryptophan abundance. Loss of these genes results in increased fungal pathogenicity and inflammation in a mouse model of aspergillosis, driven by an alternative tryptophan degradation pathway to indole derivatives and the host aryl hydrocarbon receptor. Fungal tryptophan metabolic pathways thus cooperate with the host xenobiotic response to shape host-microbe interactions in local tissue microenvironments.

Aspergillus fumigatus, One Uninucleate Species with Disparate Offspring.

Establishment of a fungal infection due to Aspergillus fumigatus relies on the efficient germination of the airborne conidia once they penetrate the respiratory tract. However, the features of conidial germination have been poorly explored and understood in this fungal species as well as in other species of filamentous fungi. We show here that the germination of A. fumigatus is asynchronous. If the nutritional environment and extensive gene deletions can modify the germination parameters for A. fumigatus, the asynchrony is maintained in all germinative conditions tested. Even though the causes for this asynchrony of conidial germination remain unknown, asynchrony is essential for the completion of the biological cycle of this filamentous fungus.

Characterization of Extracellular Vesicles Produced by Aspergillus fumigatus Protoplasts.

Extracellular vesicles (EVs) are membranous compartments produced by yeast and mycelial forms of several fungal species. One of the difficulties in perceiving the role of EVs during the fungal life, and particularly in cell wall biogenesis, is caused by the presence of a thick cell wall. One alternative to have better access to these vesicles is to use protoplasts. This approach has been investigated here with Aspergillus fumigatus, one of the most common opportunistic fungal pathogens worldwide. Analysis of regenerating protoplasts by scanning electron microscopy and fluorescence microscopy indicated the occurrence of outer membrane projections in association with surface components and the release of particles with properties resembling those of fungal EVs. EVs in culture supernatants were characterized by transmission electron microscopy and nanoparticle tracking analysis. Proteomic and glycome analysis of EVs revealed the presence of a complex array of enzymes related to lipid/sugar metabolism, pathogenic processes, and cell wall biosynthesis. Our data indicate that (i) EV production is a common feature of different morphological stages of this major fungal pathogen and (ii) protoplastic EVs are promising tools for undertaking studies of vesicle functions in fungal cells.IMPORTANCE Fungal cells use extracellular vesicles (EVs) to export biologically active molecules to the extracellular space. In this study, we used protoplasts of Aspergillus fumigatus, a major fungal pathogen, as a model to evaluate the role of EV production in cell wall biogenesis. Our results demonstrated that wall-less A. fumigatus exports plasma membrane-derived EVs containing a complex combination of proteins and glycans. Our report is the first to characterize fungal EVs in the absence of a cell wall. Our results suggest that protoplasts represent a promising model for functional studies of fungal vesicles.

Revisiting Old Questions and New Approaches to Investigate the Fungal Cell Wall Construction.

The beginning of our understanding of the cell wall construction came from the work of talented biochemists in the 70-80's. Then came the era of sequencing. Paradoxically, the accumulation of fungal genomes complicated rather than solved the mystery of cell wall construction, by revealing the involvement of a much higher number of proteins than originally thought. The situation has become even more complicated since it is now recognized that the cell wall is an organelle whose composition continuously evolves with the changes in the environment or with the age of the fungal cell. The use of new and sophisticated technologies to observe cell wall construction at an almost atomic scale should improve our knowledge of the cell wall construction. This essay will present some of the major and still unresolved questions to understand the fungal cell wall biosynthesis and some of these exciting futurist approaches.

What Are the Functions of Chitin Deacetylases in Aspergillus fumigatus?

Deacetylation of chitin by chitin deacetylases (Cda) results in the formation of chitosan. Chitosan, a polymer of ß1,4 linked glucosamine, plays multiple roles in the function of the fungal cell wall, including virulence and evasion of host immune responses. In this study, the roles of chitosan and putative CDAs in cell wall structure and virulence of Aspergillus fumigatus were investigated. Low levels of chitosan were found in the conidial and cell wall of A. fumigatus. Seven putative CDA genes were identified, disrupted and the phenotype of the single mutants and the septuple mutants were investigated. No alterations in fungal cell wall chitosan levels, changes in fungal growth or alterations in virulence were detected in the single or septuple Δcda1-7 mutant strains. Collectively, these results suggest that chitosan is a minority component of the A. fumigatus cell wall, and that the seven candidate Cda proteins do not play major roles in fungal cell wall synthesis or virulence. However, Cda2 is involved in conidiation, suggesting that this enzyme may play a role in N-acetyl-glucosamine metabolism.

Interactions between Aspergillus fumigatus and Pulmonary Bacteria: Current State of the Field, New Data, and Future Perspective.

Aspergillus fumigatus and Pseudomonas aeruginosa are central fungal and bacterial members of the pulmonary microbiota. The interactions between A. fumigatus and P. aeruginosa have only just begun to be explored. A balance between inhibitory and stimulatory effects on fungal growth was observed in mixed A. fumigatus-P. aeruginosa cultures. Negative interactions have been seen for homoserine-lactones, pyoverdine and pyochelin resulting from iron starvation and intracellular inhibitory reactive oxidant production. In contrast, several types of positive interactions were recognized. Dirhamnolipids resulted in the production of a thick fungal cell wall, allowing the fungus to resist stress. Phenazines and pyochelin favor iron uptake for the fungus. A. fumigatus is able to use bacterial volatiles to promote its growth. The immune response is also differentially regulated by co-infections.

Aspergillus fumigatus corneal infection is regulated by chitin synthases and by neutrophil-derived acidic mammalian chitinase.

Aspergillus fumigatus is an important cause of pulmonary and systemic infections in immune compromised individuals, and of corneal ulcers and blindness in immune competent patients. To examine the role of chitin synthases in Aspergillus corneal infection, we analyzed Aspergillus mutants of chitin synthase family 1 and family 2, and found that compared with the parent strain, the quadruple mutants from both families were more readily killed by neutrophils in vitro, and that both also exhibited impaired hyphal growth in the cornea. Further, inhibition of chitin synthases using Nikkomycin Z enhanced neutrophil killing in vitro and in vivo in a murine model of A. fumigatus corneal infection. Acidic mammalian chitinase (AMCase) is mostly produced by macrophages in asthmatic lungs; however, we now demonstrate that neutrophils are a major source of AMCase, which inhibits hyphal growth. In A. fumigatus corneal infection, neutrophils are the major source of AMCase, and addition of AMCase inhibitors or adoptive transfer of neutrophils from AMCase-/- mice resulted in impaired hyphal killing. Together, these findings identify chitin synthases as important fungal virulence factors and neutrophil-derived AMCase as an essential mediator of host defense.

The puzzling construction of the conidial outer layer of Aspergillus fumigatus.

If the mycelium of Aspergillus fumigatus is very short-lived in the laboratory, conidia can survive for years. This survival capacity and extreme resistance to environmental insults is a major biological characteristic of this fungal species. Moreover, conidia, which easily reach the host alveola, are the infective propagules. Earlier studies have shown the role of some molecules of the outer conidial layer in protecting the fungus against the host defense. The outer layer of the conidial cell wall, directly in contact with the host cells, consists of α-(1,3)-glucan, melanin, and proteinaceous rodlets. This study is focused on the global importance of this outer layer. Single and multiple mutants without one to three major components of the outer layer were constructed and studied. The results showed that the absence of the target molecules resulting from multiple gene deletions led to unexpected phenotypes without any logical additivity. Unexpected compensatory cell wall surface modifications were indeed observed, such as the synthesis of the mycelial virulence factor galactosaminogalactan, the increase in chitin and glycoprotein concentration or particular changes in permeability. However, sensitivity of the multiple mutants to killing by phagocytic host cells confirmed the major importance of melanin in protecting conidia.

Author Correction: Iron restriction inside macrophages regulates pulmonary host defense against Rhizopus species.

The original version of this Article contained an error in the spelling of the author Emilien Etienne, which was incorrectly given as Emilien Ettiene. These errors have now been corrected in both the PDF and HTML versions of the Article.

Chemical Synthesis and Application of Biotinylated Oligo-α-(1 → 3)-d-Glucosides To Study the Antibody and Cytokine Response against the Cell Wall α-(1 → 3)-d-Glucan of Aspergillus fumigatus.

Biotinylated hepta-, nona- and undeca-α-(1 → 3)-d-glucosides representing long oligosaccharides of α-(1 → 3)-d-glucan, one of the major components of the cell walls of the fungal pathogen Aspergillus fumigatus, were synthesized for the first time via a blockwise strategy. Convergent assembly of the α-(1 → 3)-d-glucan chains was achieved by glycosylation with oligoglucoside derivatives bearing 6- O-benzoyl groups. Those groups are capable of remote α-stereocontrolling participation, making them efficient α-directing tools even in the case of large glycosyl donors. Synthetic biotinylated oligoglucosides (and biotinylated derivatives of previously synthesized tri- and penta-α-(1 → 3)-d-glucosides) loaded on streptavidin microtiter plates were shown to be better recognized by anti-α-(1 → 3)-glucan human polyclonal antibodies and to induce higher cytokine responses upon stimulation of human peripheral blood mononuclear cells than their natural counterpart, α-(1 → 3)-d-glucan, immobilized on a conventional microtiter plate. Attachment of the synthetic oligosaccharides equipped with a hydrophilic spacer via the streptavidin-biotin pair allows better spatial presentation and control of the loading compared to the random sorption of natural α-(1 → 3)-glucan. Increase of oligoglucoside length results in their better recognition and enhancement of cytokine production. Thus, using synthetic α-(1 → 3)-glucan oligosaccharides, we developed an assay for the host immune response that is more sensitive than the assay based on native α-(1 → 3)-glucan.

Iron restriction inside macrophages regulates pulmonary host defense against Rhizopus species.

Mucormycosis is a life-threatening respiratory fungal infection predominantly caused by Rhizopus species. Mucormycosis has incompletely understood pathogenesis, particularly how abnormalities in iron metabolism compromise immune responses. Here we show how, as opposed to other filamentous fungi, Rhizopus spp. establish intracellular persistence inside alveolar macrophages (AMs). Mechanistically, lack of intracellular swelling of Rhizopus conidia results in surface retention of melanin, which induces phagosome maturation arrest through inhibition of LC3-associated phagocytosis. Intracellular inhibition of Rhizopus is an important effector mechanism, as infection of immunocompetent mice with swollen conidia, which evade phagocytosis, results in acute lethality. Concordantly, AM depletion markedly increases susceptibility to mucormycosis. Host and pathogen transcriptomics, iron supplementation studies, and genetic manipulation of iron assimilation of fungal pathways demonstrate that iron restriction inside macrophages regulates immunity against Rhizopus. Our findings shed light on the pathogenetic mechanisms of mucormycosis and reveal the role of macrophage-mediated nutritional immunity against filamentous fungi.

Calcium sequestration by fungal melanin inhibits calcium-calmodulin signalling to prevent LC3-associated phagocytosis.

LC3-associated phagocytosis (LAP) is a non-canonical autophagy pathway regulated by Rubicon, with an emerging role in immune homeostasis and antifungal host defence. Aspergillus cell wall melanin protects conidia (spores) from killing by phagocytes and promotes pathogenicity through blocking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent activation of LAP. However, the signalling regulating LAP upstream of Rubicon and the mechanism of melanin-induced inhibition of this pathway remain incompletely understood. Herein, we identify a Ca2+ signalling pathway that depends on intracellular Ca2+ sources from endoplasmic reticulum, endoplasmic reticulum-phagosome communication, Ca2+ release from phagosome lumen and calmodulin (CaM) recruitment, as a master regulator of Rubicon, the phagocyte NADPH oxidase NOX2 and other molecular components of LAP. Furthermore, we provide genetic evidence for the physiological importance of Ca2+-CaM signalling in aspergillosis. Finally, we demonstrate that Ca2+ sequestration by Aspergillus melanin inside the phagosome abrogates activation of Ca2+-CaM signalling to inhibit LAP. These findings reveal the important role of Ca2+-CaM signalling in antifungal immunity and identify an immunological function of Ca2+ binding by melanin pigments with broad physiological implications beyond fungal disease pathogenesis.

Aspergillus fumigatus Cell Wall α-(1,3)-Glucan Stimulates Regulatory T-Cell Polarization by Inducing PD-L1 Expression on Human Dendritic Cells.

Background: Human dendritic cell (DC) response to α-(1,3)-glucan polysaccharide of Aspergillus fumigatus and ensuing CD4+ T-cell polarization are poorly characterized. Methods: α-(1,3)-Glucan was isolated from A. fumigatus conidia and mycelia cell wall. For the analysis of polarization, DCs and autologous naive CD4+ T cells were cocultured. Phenotype of immune cells was analyzed by flow cytometry, and cytokines by enzyme-linked immunosorbent assay (ELISA). Blocking antibodies were used to dissect the role of Toll-like receptor 2 (TLR2) and programmed death-ligand 1 (PD-L1) in regulating α-(1,3)-glucan-mediated DC activation and T-cell responses. DCs from TLR2-deficient mice were additionally used to consolidate the findings. Results: α-(1,3)-Glucan induced the maturation of DCs and was dependent in part on TLR2. "α-(1,3)-Glucan-educated" DCs stimulated the activation of naive T cells and polarized a subset of these cells into CD4+CD25+FoxP3+ regulatory T cells (Tregs). Mechanistically, Treg stimulation by α-(1,3)-glucan was dependent on the PD-L1 pathway that negatively regulated interferon-gamma (IFN-γ) secretion. Short α-(1,3)-oligosaccharides lacked the capacity to induce maturation of DCs but significantly blocked α-(1,3)-glucan-induced Treg polarization. Conclusions: PD-L1 dictates the balance between Treg and IFN-γ responses induced by α-(1,3)-glucan. Our data provide a rationale for the exploitation of immunotherapeutic approaches that target PD-1-PD-L1 to enhance protective immune responses to A. fumigatus infections.

The Cell Wall of the Human Fungal Pathogen Aspergillus fumigatus: Biosynthesis, Organization, Immune Response, and Virulence.

More than 90% of the cell wall of the filamentous fungus Aspergillus fumigatus comprises polysaccharides. Biosynthesis of the cell wall polysaccharides is under the control of three types of enzymes: transmembrane synthases, which are anchored to the plasma membrane and use nucleotide sugars as substrates, and cell wall-associated transglycosidases and glycosyl hydrolases, which are responsible for remodeling the de novo synthesized polysaccharides and establishing the three-dimensional structure of the cell wall. For years, the cell wall was considered an inert exoskeleton of the fungal cell. The cell wall is now recognized as a living organelle, since the composition and cellular localization of the different constitutive cell wall components (especially of the outer layers) vary when the fungus senses changes in the external environment. The cell wall plays a major role during infection. The recognition of the fungal cell wall by the host is essential in the initiation of the immune response. The interactions between the different pattern-recognition receptors (PRRs) and cell wall pathogen-associated molecular patterns (PAMPs) orientate the host response toward either fungal death or growth, which would then lead to disease development. Understanding the molecular determinants of the interplay between the cell wall and host immunity is fundamental to combatting Aspergillus diseases.

Dirhamnolipids secreted from Pseudomonas aeruginosa modify anjpegungal susceptibility of Aspergillus fumigatus by inhibiting ß1,3 glucan synthase activity.

Pseudomonas aeruginosa and Aspergillus fumigatus are the two microorganisms responsible for most of the chronic infections in cystic fibrosis patients. P. aeruginosa is known to produce quorum-sensing controlled rhamnolipids during chronic infections. Here we show that the dirhamnolipids secreted from P. aeruginosa (i) induce A. fumigatus to produce an extracellular matrix, rich in galactosaminogalactan, 1,8-dihydroxynaphthalene (DHN)- and pyo-melanin, surrounding their hyphae, which facilitates P. aeruginosa binding and (ii) inhibit A. fumigatus growth by blocking ß1,3 glucan synthase (GS) activity, thus altering the cell wall architecture. A. fumigatus in the presence of diRhls resulted in a growth phenotype similar to that upon its treatment with anjpegungal echinocandins, showing multibranched hyphae and thicker cell wall rich in chitin. The diRhl structure containing two rhamnose moieties attached to fatty acyl chain is essential for the interaction with ß1,3 GS; however, the site of action of diRhls on GS is different from that of echinocandins, and showed synergistic anjpegungal effect with azoles.

Modifications to the composition of the hyphal outer layer of Aspergillus fumigatus modulates HUVEC proteins related to inflammatory and stress responses.

Aspergillus fumigatus, the main etiologic agent causing invasive aspergillosis, can induce an inflammatory response and a prothrombotic phenotype upon contact with human umbilical vein endothelial cells (HUVECs). However, the fungal molecules involved in this endothelial response remain unknown. A. fumigatus hyphae produce an extracellular matrix composed of galactomannan, galactosaminogalactan and α-(1,3)-glucan. In this study, we investigated the consequences of UGM1 gene deletion in A. fumigatus, which produces a mutant with increased galactosaminogalactan production. The ∆ugm1 mutant exhibited an HUVEC-hyperadhesive phenotype and induced increased endothelial TNF-α secretion and tissue factor mRNA overexpression in this "semi-professional" immune host cell. Using a shotgun proteomics approach, we show that the A. fumigatus ∆ugm1 strain can modulate the levels of proteins in important endothelial pathways related to the inflammatory response mediated by TNF-α and to stress response pathways. Furthermore, a purified galactosaminogalactan fraction was also able to induce TNF-α secretion and the coincident HUVEC pathways regulated by the ∆ugm1 mutant, which overexpresses this component, as demonstrated by fluorescence microscopy. This work contributes new data regarding endothelial mechanisms in response to A. fumigatus infection. SIGNIFICANCE: Invasive aspergillosis is the main opportunistic fungal infection described in neutropenic hematologic patients. One important clinical aspect of this invasive fungal infection is vascular thrombosis, which could be related, at least in part, to the activation of endothelial cells, as shown in previous reports from our group. It is known that direct contact between the A. fumigatus hyphal cell wall and the HUVEC cell surface is necessary to induce an endothelial prothrombotic phenotype and secretion of pro-inflammatory cytokines, though the cell surface components of this angioinvasive fungus that trigger this endothelial response are unknown. The present work employs a discovery-driven proteomics approach to reveal the role of one important cell wall polysaccharide of A. fumigatus, galactosaminogalactan, in the HUVEC interaction and the consequent mechanisms of endothelial activation. This is the first report of the overall panel of proteins related to the HUVEC response to a specific and purified cell wall component of the angioinvasive fungus A. fumigatus.

Role of Hydrophobins in Aspergillus fumigatus.

Resistance of Aspergillus fumigatus conidia to desiccation and their capacity to reach the alveoli are partly due to the presence of a hydrophobic layer composed of a protein from the hydrophobin family, called RodA, which covers the conidial surface. In A. fumigatus there are seven hydrophobins (RodA-RodG) belonging to class I and III. Most of them have never been studied. We constructed single and multiple hydrophobin-deletion mutants until the generation of a hydrophobin-free mutant. The phenotype, immunogenicity, and virulence of the mutants were studied. RODA is the most expressed hydrophobin in sporulating cultures, whereas RODB is upregulated in biofilm conditions and in vivo Only RodA, however, is responsible for rodlet formation, sporulation, conidial hydrophobicity, resistance to physical insult or anionic dyes, and immunological inertia of the conidia. None of the hydrophobin plays a role in biofilm formation or its hydrophobicity. RodA is the only needed hydrophobin in A. fumigatus, conditioning the structure, permeability, hydrophobicity, and immune-inertia of the cell wall surface in conidia. Moreover, the defect of rodlets on the conidial cell wall surface impacts on the drug sensitivity of the fungus.

Characteristics of Aspergillus fumigatus in Association with Stenotrophomonas maltophilia in an In Vitro Model of Mixed Biofilm.

BACKGROUND: Biofilms are communal structures of microorganisms that have long been associated with a variety of persistent infections poorly responding to conventional antibiotic or antifungal therapy. Aspergillus fumigatus fungus and Stenotrophomonas maltophilia bacteria are examples of the microorganisms that can coexist to form a biofilm especially in the respiratory tract of immunocompromised patients or cystic fibrosis patients. The aim of the present study was to develop and assess an in vitro model of a mixed biofilm associating S. maltophilia and A. fumigatus by using analytical and quantitative approaches. MATERIALS AND METHODS: An A. fumigatus strain (ATCC 13073) expressing a Green Fluorescent Protein (GFP) and an S. maltophilia strain (ATCC 13637) were used. Fungal and bacterial inocula (105 conidia/mL and 106 cells/mL, respectively) were simultaneously deposited to initiate the development of an in vitro mixed biofilm on polystyrene supports at 37°C for 24 h. The structure of the biofilm was analysed via qualitative microscopic techniques like scanning electron and transmission electron microscopy, and fluorescence microscopy, and by quantitative techniques including qPCR and crystal violet staining. RESULTS: Analytic methods revealed typical structures of biofilm with production of an extracellular matrix (ECM) enclosing fungal hyphae and bacteria. Quantitative methods showed a decrease of A. fumigatus growth and ECM production in the mixed biofilm with antibiosis effect of the bacteria on the fungi seen as abortive hyphae, limited hyphal growth, fewer conidia, and thicker fungal cell walls. CONCLUSION: For the first time, a mixed A. fumigatus-S. maltophilia biofilm was validated by various analytical and quantitative approaches and the bacterial antibiosis effect on the fungus was demonstrated. The mixed biofilm model is an interesting experimentation field to evaluate efficiency of antimicrobial agents and to analyse the interactions between the biofilm and the airways epithelium.