Galactosaminogalactan activates the inflammasome to provide host protection.

Inflammasomes are important sentinels of innate immune defence that are activated in response to diverse stimuli, including pathogen-associated molecular patterns (PAMPs)1. Activation of the inflammasome provides host defence against aspergillosis2,3, which is a major health concern for patients who are immunocompromised. However, the Aspergillus fumigatus PAMPs that are responsible for inflammasome activation are not known. Here we show that the polysaccharide galactosaminogalactan (GAG) of A. fumigatus is a PAMP that activates the NLRP3 inflammasome. The binding of GAG to ribosomal proteins inhibited cellular translation machinery, and thus activated the NLRP3 inflammasome. The galactosamine moiety bound to ribosomal proteins and blocked cellular translation, which triggered activation of the NLRP3 inflammasome. In mice, a GAG-deficient Aspergillus mutant (Δgt4c) did not elicit protective activation of the inflammasome, and this strain exhibited enhanced virulence. Moreover, administration of GAG protected mice from colitis induced by dextran sulfate sodium in an inflammasome-dependent manner. Thus, ribosomes connect the sensing of this fungal PAMP to the activation of an innate immune response.

MybA, a transcription factor involved in conidiation and conidial viability of the human pathogen Aspergillus fumigatus.

Aspergillus fumigatus, a ubiquitous human fungal pathogen, produces asexual spores (conidia), which are the main mode of propagation, survival and infection of this human pathogen. In this study, we present the molecular characterization of a novel regulator of conidiogenesis and conidial survival called MybA because the predicted protein contains a Myb DNA binding motif. Cellular localization of the MybA::Gfp fusion and immunoprecipitation of the MybA::Gfp or MybA::3xHa protein showed that MybA is localized to the nucleus. RNA sequencing data and a uidA reporter assay indicated that the MybA protein functions upstream of wetA, vosA and velB, the key regulators involved in conidial maturation. The deletion of mybA resulted in a very significant reduction in the number and viability of conidia. As a consequence, the ΔmybA strain has a reduced virulence in an experimental murine model of aspergillosis. RNA-sequencing and biochemical studies of the ΔmybA strain suggested that MybA protein controls the expression of enzymes involved in trehalose biosynthesis as well as other cell wall and membrane-associated proteins and ROS scavenging enzymes. In summary, MybA protein is a new key regulator of conidiogenesis and conidial maturation and survival, and plays a crucial role in propagation and virulence of A. fumigatus.

Galactosaminogalactan of Aspergillus fumigatus, a bioactive fungal polymer.

Galactosaminogalactan (GAG) is an extracellular polysaccharide produced by the mycelium of the opportunistic human fungal pathogen Aspergillus fumigatus GAG is the first polysaccharide described as a virulence factor in medical mycology. This review presents our current knowledge of the structural organization and biosynthesis of this polymer. The function of this molecule as an adhesin that also masks Aspergillus PAMPs and the impact of GAG on the modulation of the host immune response by inducing neutropenia and blocking the IL-1 signaling pathway also will be emphasised.

Deciphering the role of the chitin synthase families 1 and 2 in the in vivo and in vitro growth of Aspergillus fumigatus by multiple gene targeting deletion.

Although chitin is an essential component of the fungal cell wall (CW), its biosynthesis and role in virulence is poorly understood. In Aspergillus fumigatus, there are eight chitin synthase (CHS) genes belonging to two families CHSA-C, CHSG in family 1 and CHSF, CHSD, CSMA, CSMB in family 2). To understand the function of these CHS genes, their single and multiple deletions were performed using ß-rec/six system to be able to delete all genes within each family (up to a quadruple ΔchsA/C/B/G mutant in family 1 and a quadruple ΔcsmA/csmB/F/D mutant in family 2). Radial growth, conidiation, mycelial/conidial morphology, CW polysaccharide content, Chs-activity, susceptibility to antifungal molecules and pathogenicity in experimental animal aspergillosis were analysed for all the mutants. Among the family 1 CHS, ΔchsA, ΔchsB and ΔchsC mutants showed limited impact on chitin synthesis. In contrast, there was reduced conidiation, altered mycelial morphotype and reduced growth and Chs-activity in the ΔchsG and ΔchsA/C/B/G mutants. In spite of this altered phenotype, these two mutants were as virulent as the parental strain in the experimental aspergillosis models. Among family 2 CHS, phenotypic defects mainly resulted from the CSMA deletion. Despite significant morphological mycelial and conidial growth phenotypes in the quadruple ΔcsmA/csmB/F/D mutant, the chitin content was poorly affected by gene deletions in this family. However, the entire mycelial cell wall structure was disorganized in the family 2 mutants that may be related to the reduced pathogenicity of the quadruple ΔcsmA/csmB/F/D mutant strain compared to the parental strain, in vivo. Deletion of the genes encompassing the two families (ΔcsmA/csmB/F/G) showed that in spite of being originated from an ancient divergence of fungi, these two families work cooperatively to synthesize chitin in A. fumigatus and demonstrate the essentiality of chitin biosynthesis for vegetative growth, resistance to antifungal drugs, and virulence of this filamentous fungus.

The protein phosphatase PhzA of A. fumigatus is involved in oxidative stress tolerance and fungal virulence.

Protein phosphatases Z that are unique to the fungal kingdom have been associated to resistance to high salt concentration, cell wall integrity, cell cycle regulation, and oxidative stress in fungi. In Aspergillus fumigatus, it was shown that PHZA is under the control of the transcription factor Skn7 and is only involved in the control of the oxidative stress. Accordingly, the ΔphzA mutant showed a defect in virulence in an experimental model of corneal infection in immunocompetent animals and that the impact on susceptibility to cell wall drugs is only secondary.

Conidium Specific Polysaccharides in Aspergillus fumigatus.

Earlier studies have shown that the outer layers of the conidial and mycelial cell walls of Aspergillus fumigatus are different. In this work, we analyzed the polysaccharidome of the resting conidial cell wall and observed major differences within the mycelium cell wall. Mainly, the conidia cell wall was characterized by (i) a smaller amount of α-(1,3)-glucan and chitin; (ii) a larger amount of ß-(1,3)-glucan, which was divided into alkali-insoluble and water-soluble fractions, and (iii) the existence of a specific mannan with side chains containing galactopyranose, glucose, and N-acetylglucosamine residues. An analysis of A. fumigatus cell wall gene mutants suggested that members of the fungal GH-72 transglycosylase family play a crucial role in the conidia cell wall ß-(1,3)-glucan organization and that α-(1,6)-mannosyltransferases of GT-32 and GT-62 families are essential to the polymerization of the conidium-associated cell wall mannan. This specific mannan and the well-known galactomannan follow two independent biosynthetic pathways.

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.

The Glycosylphosphatidylinositol-Anchored DFG Family Is Essential for the Insertion of Galactomannan into the ß-(1,3)-Glucan-Chitin Core of the Cell Wall of Aspergillus fumigatus.

The fungal cell wall is a complex and dynamic entity essential for the development of fungi. It is composed mainly of polysaccharides that are synthetized by protein complexes. At the cell wall level, enzyme activities are involved in postsynthesis polysaccharide modifications such as cleavage, elongation, branching, and cross-linking. Glycosylphosphatidylinositol (GPI)-anchored proteins have been shown to participate in cell wall biosynthesis and specifically in polysaccharide remodeling. Among these proteins, the DFG family plays an essential role in controlling polar growth in yeast. In the filamentous fungus and opportunistic human pathogen Aspergillus fumigatus, the DFG gene family contains seven orthologous DFG genes among which only six are expressed under in vitro growth conditions. Deletions of single DFG genes revealed that DFG3 plays the most important morphogenetic role in this gene family. A sextuple-deletion mutant resulting from the deletion of all in vitro expressed DFG genes did not contain galactomannan in the cell wall and has severe growth defects. This study has shown that DFG members are absolutely necessary for the insertion of galactomannan into the cell wall of A. fumigatus and that the proper cell wall localization of the galactomannan is essential for correct fungal morphogenesis in A. fumigatusIMPORTANCE The fungal cell wall is a complex and dynamic entity essential for the development of fungi. It is composed mainly of polysaccharides that are synthetized by protein complexes. Enzymes involved in postsynthesis polysaccharide modifications, such as cleavage, elongation, branching, and cross-linking, are essential for fungal life. Here, we investigated in Aspergillus fumigatus the role of the members of the Dfg family, one of the 4 GPI-anchored protein families common to yeast and molds involved in cell wall remodeling. Molecular and biochemical approaches showed that DFG members are required for filamentous growth, conidiation, and cell wall organization and are essential for the life of this fungal pathogen.

The pH regulator PacC: a host-dependent virulence factor in Botrytis cinerea.

The phytopathogenic fungus Botrytis cinerea is able to infect a wide variety of plants and plant tissues with differing chemical compositions. During its interaction with the host, this pathogen modulates its ambient pH by secreting acids or ammonia. In this work, we examined the Pal/Pac pathway, the fungal ambient pH-responsive signalling circuit, and investigated the role of the PacC transcription factor. Characterization of the BcpacC deletion mutant revealed an alteration of both fungal growth and virulence depending on the pH of the culture medium or of the host tissue. The pathogenicity of the mutant was altered on plants exhibiting a neutral pH and not on plants with acidic tissues. The capacity of the mutant to acidify its environment and, more particularly, to produce oxalic acid was affected, as was production of reactive oxygen species. Finally, proteomic profiling of the mutant secretome revealed significant changes in plant cell wall polysaccharides proteins and lipid degradation and oxidoreduction, highlighting the importance of BcPacC in the necrotrophic lifestyle of B. cinerea.

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.

Aspergillus Cell Wall Melanin Blocks LC3-Associated Phagocytosis to Promote Pathogenicity.

Concealing pathogen-associated molecular patterns (PAMPs) is a principal strategy used by fungi to avoid immune recognition. Surface exposure of PAMPs during germination can leave the pathogen vulnerable. Accordingly, ß-glucan surface exposure during Aspergillus fumigatus germination activates an Atg5-dependent autophagy pathway termed LC3-associated phagocytosis (LAP), which promotes fungal killing. We found that LAP activation also requires the genetic, biochemical or biological (germination) removal of A. fumigatus cell wall melanin. The attenuated virulence of melanin-deficient A. fumigatus is restored in Atg5-deficient macrophages and in mice upon conditional inactivation of Atg5 in hematopoietic cells. Mechanistically, Aspergillus melanin inhibits NADPH oxidase-dependent activation of LAP by excluding the p22phox subunit from the phagosome. Thus, two events that occur concomitantly during germination of airborne fungi, surface exposure of PAMPs and melanin removal, are necessary for LAP activation and fungal killing. LAP blockade is a general property of melanin pigments, a finding with broad physiological implications.

Investigation of Aspergillus fumigatus biofilm formation by various "omics" approaches.

In the lung, Aspergillus fumigatus usually forms a dense colony of filaments embedded in a polymeric extracellular matrix called biofilm (BF). This extracellular matrix embeds and glues hyphae together and protects the fungus from an outside hostile environment. This extracellular matrix is absent in fungal colonies grown under classical liquid shake conditions (PL), which were historically used to understand A. fumigatus pathobiology. Recent works have shown that the fungus in this aerial grown BF-like state exhibits reduced susceptibility to antifungal drugs and undergoes major metabolic changes that are thought to be associated to virulence. These differences in pathological and physiological characteristics between BF and liquid shake conditions suggest that the PL condition is a poor in vitro disease model. In the laboratory, A. fumigatus mycelium embedded by the extracellular matrix can be produced in vitro in aerial condition using an agar-based medium. To provide a global and accurate understanding of A. fumigatus in vitro BF growth, we utilized microarray, RNA-sequencing, and proteomic analysis to compare the global gene and protein expression profiles of A. fumigatus grown under BF and PL conditions. In this review, we will present the different signatures obtained with these three "omics" methods. We will discuss the advantages and limitations of each method and their complementarity.