FLO1 is a variable green beard gene that drives biofilm-like cooperation in budding yeast.

The budding yeast, Saccharomyces cerevisiae, has emerged as an archetype of eukaryotic cell biology. Here we show that S. cerevisiae is also a model for the evolution of cooperative behavior by revisiting flocculation, a self-adherence phenotype lacking in most laboratory strains. Expression of the gene FLO1 in the laboratory strain S288C restores flocculation, an altered physiological state, reminiscent of bacterial biofilms. Flocculation protects the FLO1 expressing cells from multiple stresses, including antimicrobials and ethanol. Furthermore, FLO1(+) cells avoid exploitation by nonexpressing flo1 cells by self/non-self recognition: FLO1(+) cells preferentially stick to one another, regardless of genetic relatedness across the rest of the genome. Flocculation, therefore, is driven by one of a few known "green beard genes," which direct cooperation toward other carriers of the same gene. Moreover, FLO1 is highly variable among strains both in expression and in sequence, suggesting that flocculation in S. cerevisiae is a dynamic, rapidly evolving social trait.

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.

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.

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.

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.

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.

Biosynthesis of cell wall mannan in the conidium and the mycelium of Aspergillus fumigatus.

The galactomannan is a major cell wall molecule of Aspergillus fumigatus. This molecule is composed of a linear mannan with a repeating unit composed of four α1,6 and α1,2 linked mannose with side chains of galactofuran. To obtain a better understanding of the mannan biosynthesis in A. fumigatus, it was decided to undertake the successive deletion of the 11 genes which are putative orthologs of the mannosyltransferases responsible for establishing α1,6 and α1,2 mannose linkages in yeast. These deletions did not lead to a reduction of the mannan content of the cell wall of the mycelium of A. fumigatus. In contrast, the mannan content of the conidial cell wall was reduced and this reduction was associated with a partial disorganization of the cell wall leading to defects in conidial survival both in vitro and in vivo.

Deletion of the α-(1,3)-glucan synthase genes induces a restructuring of the conidial cell wall responsible for the avirulence of Aspergillus fumigatus.

α-(1,3)-Glucan is a major component of the cell wall of Aspergillus fumigatus, an opportunistic human fungal pathogen. There are three genes (AGS1, AGS2 and AGS3) controlling the biosynthesis of α-(1,3)-glucan in this fungal species. Deletion of all the three AGS genes resulted in a triple mutant that was devoid of α-(1,3)-glucan in its cell wall; however, its growth and germination was identical to that of the parental strain in vitro. In the experimental murine aspergillosis model, this mutant was less pathogenic than the parental strain. The AGS deletion resulted in an extensive structural modification of the conidial cell wall, especially conidial surface where the rodlet layer was covered by an amorphous glycoprotein matrix. This surface modification was responsible for viability reduction of conidia in vivo, which explains decrease in the virulence of triple agsΔ mutant.

Aspergillus galactosaminogalactan mediates adherence to host constituents and conceals hyphal ß-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 ß-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.

SUN proteins belong to a novel family of ß-(1,3)-glucan-modifying enzymes involved in fungal morphogenesis.

BACKGROUND: SUN proteins are involved in yeast morphogenesis, but their function is unknown. RESULTS: SUN protein plays a role in the Aspergillus fumigatus morphogenesis. Biochemical properties of recombinant SUN proteins were elucidated. CONCLUSION: Both Candida albicans and Aspergillus fumigatus sun proteins show a ß-(1,3)-glucanase activity. SIGNIFICANCE: The mode of action of SUN proteins on ß-(1,3)-glucan is unique, new, and original. In yeasts, the family of SUN proteins has been involved in cell wall biogenesis. Here, we report the characterization of SUN proteins in a filamentous fungus, Aspergillus fumigatus. The function of the two A. fumigatus SUN genes was investigated by combining reverse genetics and biochemistry. During conidial swelling and mycelial growth, the expression of AfSUN1 was strongly induced, whereas the expression of AfSUN2 was not detectable. Deletion of AfSUN1 negatively affected hyphal growth and conidiation. A closer examination of the morphological defects revealed swollen hyphae, leaky tips, intrahyphal growth, and double cell wall, suggesting that, like in yeast, AfSun1p is associated with cell wall biogenesis. In contrast to AfSUN1, deletion of AfSUN2 either in the parental strain or in the AfSUN1 single mutant strain did not affect colony and hyphal morphology. Biochemical characterization of the recombinant AfSun1p and Candida albicans Sun41p showed that both proteins had a unique hydrolysis pattern: acting on ß-(1,3)-oligomers from dimer up to insoluble ß-(1,3)-glucan. Referring to the CAZy database, it is clear that fungal SUN proteins represent a new family of glucan hydrolases (GH132) and play an important morphogenetic role in fungal cell wall biogenesis and septation.

Surface structure characterization of Aspergillus fumigatus conidia mutated in the melanin synthesis pathway and their human cellular immune response.

In Aspergillus fumigatus, the conidial surface contains dihydroxynaphthalene (DHN)-melanin. Six-clustered gene products have been identified that mediate sequential catalysis of DHN-melanin biosynthesis. Melanin thus produced is known to be a virulence factor, protecting the fungus from the host defense mechanisms. In the present study, individual deletion of the genes involved in the initial three steps of melanin biosynthesis resulted in an altered conidial surface with masked surface rodlet layer, leaky cell wall allowing the deposition of proteins on the cell surface and exposing the otherwise-masked cell wall polysaccharides at the surface. Melanin as such was immunologically inert; however, deletion mutant conidia with modified surfaces could activate human dendritic cells and the subsequent cytokine production in contrast to the wild-type conidia. Cell surface defects were rectified in the conidia mutated in downstream melanin biosynthetic pathway, and maximum immune inertness was observed upon synthesis of vermelone onward. These observations suggest that although melanin as such is an immunologically inert material, it confers virulence by facilitating proper formation of the A. fumigatus conidial surface.

Aspergillus cell wall and biofilm.

The fungal cell is surrounded by a cell wall that acts as a sieve and a reservoir for effector molecules that play an active role during infection. This cell wall is essential for fungal growth as well as for resisting host defense mechanisms. The Aspergillus fumigatus cell wall is almost exclusively composed of polysaccharides. The fibrillar core is composed of a branched ß-(1,3)-glucan to which chitin, ß-(1,3)-/ß-(1,4)-glucan, and galactomannan are covalently bound. The alkali-soluble amorphous fraction is mainly composed of α-(1,3)-glucan that has adhesive property and stabilizes the cell wall. Although the same polysaccharides are found in the cell wall of different A. fumigatus morphotypes (conidia and hyphae), their concentration and localization are different. Conidial (the morphotype that mainly enters host respiratory system) cell wall is covered by an outer layer of rodlets and melanin, which confers hydrophobic properties and imparts immunological inertness. In contrast, outer layer of the hypha contains galactosaminogalactan, recently identified as an A. fumigatus virulence factor. The hypha grows either as a network of agglutinated and hydrophobic mass (called mycelium) embedded in an extracellular matrix (ECM) rich in polysaccharides, hydrophobin, and melanin or segregated without ECM.

α1,3 glucans are dispensable in Aspergillus fumigatus.

A triple α1,3 glucan synthase mutant of Aspergillus fumigatus obtained by successive deletions of the three α1,3 glucan synthase genes (AGS1, AGS2, and AGS3) has a cell wall devoid of α1,3 glucans. The lack of α1,3 glucans affects neither conidial germination nor mycelial vegetative growth and is compensated by an increase in ß1,3 glucan and/or chitin content.

Global transcriptome changes underlying colony growth in the opportunistic human pathogen Aspergillus fumigatus.

Aspergillus fumigatus is the most common and deadly pulmonary fungal infection worldwide. In the lung, the fungus usually forms a dense colony of filaments embedded in a polymeric extracellular matrix. To identify candidate genes involved in this biofilm (BF) growth, we used RNA-Seq to compare the transcriptomes of BF and liquid plankton (PL) growth. Sequencing and mapping of tens of millions sequence reads against the A. fumigatus transcriptome identified 3,728 differentially regulated genes in the two conditions. Although many of these genes, including the ones coding for transcription factors, stress response, the ribosome, and the translation machinery, likely reflect the different growth demands in the two conditions, our experiment also identified hundreds of candidate genes for the observed differences in morphology and pathobiology between BF and PL. We found an overrepresentation of upregulated genes in transport, secondary metabolism, and cell wall and surface functions. Furthermore, upregulated genes showed significant spatial structure across the A. fumigatus genome; they were more likely to occur in subtelomeric regions and colocalized in 27 genomic neighborhoods, many of which overlapped with known or candidate secondary metabolism gene clusters. We also identified 1,164 genes that were downregulated. This gene set was not spatially structured across the genome and was overrepresented in genes participating in primary metabolic functions, including carbon and amino acid metabolism. These results add valuable insight into the genetics of biofilm formation in A. fumigatus and other filamentous fungi and identify many relevant, in the context of biofilm biology, candidate genes for downstream functional experiments.

The composition of the culture medium influences the ß-1,3-glucan metabolism of Aspergillus fumigatus and the antifungal activity of inhibitors of ß-1,3-glucan synthesis.

In vitro testing of Aspergillus fumigatus susceptibility to echinocandins has always been a challenge. Using a simple and quick colorimetric method to analyze the activity of inhibitors of ß-1,3-glucan synthesis, we found that the composition of the culture medium significantly influences glucan synthesis and consequently the antifungal properties of inhibitors of ß-1,3-glucan synthesis when they are tested alone or in combination with chitin synthase inhibitors.

Chitin synthases with a myosin motor-like domain control the resistance of Aspergillus fumigatus to echinocandins.

Aspergillus fumigatus has two chitin synthases (CSMA and CSMB) with a myosin motor-like domain (MMD) arranged in a head-to-head configuration. To understand the function of these chitin synthases, single and double csm mutant strains were constructed and analyzed. Although there was a slight reduction in mycelial growth of the mutants, the total chitin synthase activity and the cell wall chitin content were similar in the mycelium of all of the mutants and the parental strain. In the conidia, chitin content in the ΔcsmA strain cell wall was less than half the amount found in the parental strain. In contrast, the ΔcsmB mutant strain and, unexpectedly, the ΔcsmA/ΔcsmB mutant strain did not show any modification of chitin content in their conidial cell walls. In contrast to the hydrophobic conidia of the parental strain, conidia of all of the csm mutants were hydrophilic due to the presence of an amorphous material covering the hydrophobic surface-rodlet layer. The deletion of CSM genes also resulted in an increased susceptibility of resting and germinating conidia to echinocandins. These results show that the deletion of the CSMA and CSMB genes induced a significant disorganization of the cell wall structure, even though they contribute only weakly to the overall cell wall chitin synthesis.

Members of protein O-mannosyltransferase family in Aspergillus fumigatus differentially affect growth, morphogenesis and viability.

O-mannosylation is an essential protein modification in eukaryotes. It is initiated at the endoplasmic reticulum by O-mannosyltransferases (PMT) that are evolutionary conserved from yeast to humans. The PMT family is phylogenetically classified into PMT1, PMT2 and PMT4 subfamilies, which differ in protein substrate specificity and number of genes per subfamily. In this study, we characterized for the first time the whole PMT family of a pathogenic filamentous fungus, Aspergillus fumigatus. Genome analysis showed that only one member of each subfamily is present in A. fumigatus, PMT1, PMT2 and PMT4. Despite the fact that all PMTs are transmembrane proteins with conserved peptide motifs, the phenotype of each PMT deletion mutant was very different in A. fumigatus. If disruption of PMT1 did not reveal any phenotype, deletion of PMT2 was lethal. Disruption of PMT4 resulted in abnormal mycelial growth and highly reduced conidiation associated to significant proteomic changes. The double pmt1pmt4 mutant was lethal. The single pmt4 mutant exhibited an exquisite sensitivity to echinocandins that is associated to major changes in the expression of signal transduction cascade genes. These results indicate that the PMT family members play a major role in growth, morphogenesis and viability of A. fumigatus.

In vivo biofilm composition of Aspergillus fumigatus.

The in vivo composition of the mycelial extracellular matrix (ECM) of Aspergillus fumigatus during host invasion is reported here for the first time. A new galactosaminogalactan and the galactomannan were the major polysaccharides of the in vivo ECM. The composition of the ECM in vivo varied with the aspergillosis pathologies.

Aspergillus fumigatus biofilms in the clinical setting.

We discuss in this work the role of Aspergillus biofilms in the clinical setting by reviewing the most recent findings on this topic. Aspergillus fumigatus can produce in vitro an extracellular hydrophobic matrix with typical biofilm characteristics under all static conditions tested, i.e., agar media, polystyrene and bronchial epithelial cells. Under static conditions the mycelial growth is greater than in shaken, submerged conditions. The extracellular matrix (ECM) is composed of galactomannan, α-1,3-glucans, monosaccharides and polyols, melanin and proteins including major antigens and hydrophobins. Typical biofilm structures were observed in the aspergillomas from two patients and in a murine model of invasive pulmonary aspergillosis. The results indicate that α-1,3-glucans plays a predominant role in the agglutination of the hyphae together in aerial conditions, and that nutrient starvation was responsible for mycelial death in aspergilloma. Melanin was produced during the infection, suggesting that this pigment is necessary for lung tissue invasion. All antifungal drugs are significantly less effective when A. fumigatus is grown under biofilm vs. planktonic conditions. Chronic persistence of a unique genotype of A. fumigatus in the respiratory tract of CF-patients and the presence of an ECM in vivo may have some therapeutical application for aspergillosis. The most appropriate antifungal drug should not be selected only on the basis of its efficiency to kill in vitro grown fungal cells, but also on its ability to penetrate the ECM.