Anne Beauvais

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.

Extracellular vesicles (EVs) are outer membranous compartments produced by yeast and mycelial forms of several fungal species. One of the difficulties to perceive the role of EVs during the fungal life is the fact that an active secretion of these EVs has not been clearly demonstrated in situ due to the presence of a thick cell wall. One alternative to have a 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 a promising tool to undertake studies of vesicle functions in fungal cells. IMPORTANCE: Fungal cells use extracellular vesicles (EVs) to export biologically active molecules to the outer space. Since fungal cells are encaged in a thick cell wall, it is reasonable to expect that this structure might impact the vesicle-mediated molecular export. In this study, we used protoplasts of Aspergillus fumigatus , a major fungal pathogen, as a model to evaluate EV production in the absence of a cell wall. Our results demonstrated that wall-less A. fumigatus exports plasma membrane-derived EVs containing a complex combination of proteins and glycans. Our study is the first to characterize fungal EVs in the absence of a cell wall. Our results suggest that protoplasts are a promising model for functional studies of fungal vesicles.

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.

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.

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.

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.

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 β1,3 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 β1,3 GS; however, the site of action of diRhls on GS is different from that of echinocandins, and showed synergistic anjpegungal effect with azoles.

In vivo, Aspergillus fumigatus grows as a typical biofilm with hyphae covered by an extracellular matrix (ECM) composed of polysaccharides, galactomannan, and galactosaminogalactan. α1,3 glucans and melanin are also constitutive of the ECM in aspergilloma but not in invasive aspergillosis. In vitro , two biofilm models were established to mimic the in vivo situation. The first model (model 1) uses submerged liquid conditions and is characterized by slow growth, while the second model (model 2) uses agar medium and aerial conditions and is characterized by rapid growth. The composition of the ECM was studied only in the second model and has been shown to be composed of galactomannan, galactosaminogalactan (GAG), and α1,3 glucans, melanin, antigens, and hydrophobins. The presence of extracellular DNA was detected in model 1 biofilm but not in model 2. Transcriptomic analysis employing both biofilm models showed upregulation of genes coding for proteins involved in the biosynthesis of secondary metabolites, adhesion, and drug resistance. However, most data on A. fumigatus biofilms have been obtained in vitro and should be confirmed using in vivo animal models. There is a need for new therapeutic antibiofilm strategies that focus on the use of combination therapy, since biofilm formation poses an important clinical problem due to their resistance to antifungal agents. Furthermore, in vivo investigations of A. fumigatus biofilms that incorporate the associated microbiota are needed. Such studies will add another layer of complexity to our understanding of the role of A. fumigatus biofilm during lung invasion.

The opportunistic fungal pathogen Aspergillus fumigatus is increasingly found as a coinfecting agent along with Pseudomonas aeruginosa in cystic fibrosis patients. Amongst the numerous molecules secreted by P. aeruginosa during its growth, phenazines constitute a major class. P. aeruginosa usually secreted four phenazines, pyocyanin (PYO), phenazine-1-carboxamide (PCN), 1-hydroxyphenazine (1-HP) and phenazine-1-carboxylic acid (PCA). These phenazines inhibited the growth of A. fumigatus but the underlying mechanisms and the impact of these four phenazines on A. fumigatus biology were not known. In the present study, we analyzed the functions of the four phenazines and their mode of action on A. fumigatus. All four phenazines showed A. fumigatus growth inhibitory effects by inducing production of reactive oxygen species (ROS), specifically O₂^·− and reactive nitrogen species (RNS), ONOO⁻. A. fumigatus Sod2p was the major factor involved in resistance against the ROS and RNS induced by phenazines. Sub-inhibitory concentrations of PYO, PCA and PCN promote A. fumigatus growth by an independent iron-uptake acquisition. Of the four phenazines 1-HP had a redox-independent function; being able to chelate metal ions 1-HP induced A. fumigatus iron starvation. Our data show the fine-interactions existing between A. fumigatus and P. aeruginosa, which can lead to stimulatory or antagonistic effects.