Isabelle Mouyna

Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus.

Aspergillus fumigatus is exceptional among microorganisms in being both a primary and opportunistic pathogen as well as a major allergen. Its conidia production is prolific, and so human respiratory tract exposure is almost constant. A. fumigatus is isolated from human habitats and vegetable compost heaps. In immunocompromised individuals, the incidence of invasive infection can be as high as 50% and the mortality rate is often about 50% (ref. 2). The interaction of A. fumigatus and other airborne fungi with the immune system is increasingly linked to severe asthma and sinusitis. Although the burden of invasive disease caused by A. fumigatus is substantial, the basic biology of the organism is mostly obscure. Here we show the complete 29.4-megabase genome sequence of the clinical isolate Af293, which consists of eight chromosomes containing 9,926 predicted genes. Microarray analysis revealed temperature-dependent expression of distinct sets of genes, as well as 700 A. fumigatus genes not present or significantly diverged in the closely related sexual species Neosartorya fischeri, many of which may have roles in the pathogenicity phenotype. The Af293 genome sequence provides an unparalleled resource for the future understanding of this remarkable fungus.

Specific molecular features in the organization and biosynthesis of the cell wall of Aspergillus fumigatus

The cell wall of Aspergillus fumigatus is composed of a branched beta1,3 glucan covalently bound to chitin, beta1,3, beta1,4 glucans, and galactomannan, that is embedded in an amorphous cement composed of alpha1,3 glucan, galactomannan and polygalactosamin. The mycelial cell wall of A. fumigatus is very different from the yeast Saccharomyces cerevisiae cell wall, and in particular lacks beta1,6 glucans and proteins covalently bound to cell wall polysaccharides. The differences in cell wall composition between the mould A. fumigatus and the yeast S. cerevisiae are also reflected at the genomic level where unique features have been identified in A. fumigatus. A single gene codes for the glucan synthase catalytic subumit; this finding has lead to the development of a RNAi methodology for the disruption of essential genes in A. fumigatus. In contrast to the glucan synthase, multiple genes have been found in the chitin synthase and the alpha glucan synthase families; in spite of homologous sequences, each gene in each family have very different function. Similarly homologous mannosyltransferase genes are found in yeast and moulds but they lead to the synthesis of very different N-mannan structures. This chemo-genomic comparative analysis has also suggested that GPI-anchored proteins do not have a role of linker in the three dimensional organization of the fungal cell wall.

Deletion of GEL2 encoding for a β(1-3)glucanosyltransferase affects morphogenesis and virulence in Aspergillus fumigatus

The first fungal glycosylphosphatidylinositol an‐chored β(1–3)glucanosyltranferase (Gel1p) has been described in Aspergillus fumigatus and its encoding gene GEL1 identified. Glycosylphosphatidylinositol‐anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall. We characterize here GEL2, a homologue of GEL1. Both homologues share common characteristics: (i) GEL1 and GEL2 are constitutively expressed during over a range of growth conditions; (ii) Gel2p is also a putative GPI‐anchored protein and shares the same β(1–3)glucanosyltransferase activity as Gel1p and (iii) GEL2, like GEL1, is able to complement the Δgas1 deletion in Saccharomyces cerevisiae confirming that Gelp and Gasp have the same enzymatic activity. However, disruption of GEL1 did not result in a phenotype whereas a Δgel2 mutant and the double mutant Δgel1Δgel2 exhibit slower growth, abnormal conidiogenesis, and an altered cell wall composition. In addition, the Δgel2 and the Δgel1Δgel2 mutant have reduced virulence in a murine model of invasive aspergillosis. These data suggest for the first time that β(1–3)glucanosyltransferase activity is required for both morphogenesis and virulence in A. fumigatus.

A 1,3-beta-glucanosyltransferase isolated from the cell wall of Aspergillus fumigatus is a homologue of the yeast Bgl2p.

A 1,3-beta-glucanosyltransferase which introduces intrachain 1,6-beta linkages into 1,3-beta-glucan was isolated from the cell wall of Aspergillus fumigatus. The biochemical and molecular characterization of the A. fumigatus transferase showed it was homologous to the Saccharomyces cerevisiae and Candida albicans transferase Bgl2p. A null mutant constructed in A. fumigatus by gene replacement did not show a distinct phenotype from the parental strain. The putative function of this major cell-wall-associated protein is discussed.

Identification of the catalytic residues of the first family of beta(1-3)glucanosyltransferases identified in fungi.

A new family of glycosylphosphatidylinositol-anchored beta(1-3)glucanosyltransferases (Gelp), recently identified and characterized in the filamentous fungus Aspergillus fumigatus, showed functional similarity to the Gas/Phr/Epd protein families, which are involved in yeast morphogenesis. Sequence comparisons and hydrophobic cluster analysis (HCA) showed that all the Gas/Phr/Epd/Gel proteins belong to a new family of glycosylhydrolases, family 72. We confirmed by site-directed mutagenesis and biochemical analysis that the two conserved glutamate residues (the putative catalytic residues of this family, as determined by HCA) are involved in the active site of this family of glycosylhydrolases.

Characterization of a cell-wall acid phosphatase (PhoAp) in Aspergillus fumigatus

In the filamentous fungus Aspergillus fumigatus, the vast majority of the cell-wall-associated proteins are secreted proteins that are in transit in the cell wall. These proteins can be solubilized by detergents and reducing agents. Incubation of a SDS/beta-mercaptoethanol-treated cell-wall extract with various recombinant enzymes that hydrolyse cell-wall polysaccharides resulted in the release of a unique protein in minute amounts only after incubation of the cell wall in the presence of 1,3-beta-glucanase. Sequence analysis and biochemical studies showed that this glycoprotein, with an apparent molecular mass of 80 kDa, was an acid phosphatase (PhoAp) that was active on both phosphate monoesters and phosphate diesters. PhoAp is a glycosylphosphatidylinositol-anchored protein that was recovered in the culture filtrate and cell-wall fraction of A. fumigatus after cleavage of its anchor. It is also a phosphate-repressible acid phosphatase. The absence of PhoAp from a phosphate-rich medium was not associated with a reduction in fungal growth, indicating that this cell-wall-associated protein does not play a role in the morphogenesis of A. fumigatus.

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.

Molecular Mechanisms of Yeast Cell Wall Glucan Remodeling

Yeast cell wall remodeling is controlled by the equilibrium between glycoside hydrolases, glycosyltransferases, and transglycosylases. Family 72 glycoside hydrolases (GH72) are ubiquitous in fungal organisms and are known to possess significant transglycosylase activity, producing elongated β(1–3) glucan chains. However, the molecular mechanisms that control the balance between hydrolysis and transglycosylation in these enzymes are not understood. Here we present the first crystal structure of a glucan transglycosylase, Saccharomyces cerevisiae Gas2 (ScGas2), revealing a multidomain fold, with a (βα)8 catalytic core and a separate glucan binding domain with an elongated, conserved glucan binding groove. Structures of ScGas2 complexes with different β-glucan substrate/product oligosaccharides provide “snapshots” of substrate binding and hydrolysis/transglycosylation giving the first insights into the mechanisms these enzymes employ to drive β(1–3) glucan elongation. Together with mutagenesis and analysis of reaction products, the structures suggest a “base occlusion” mechanism through which these enzymes protect the covalent protein-enzyme intermediate from a water nucleophile, thus controlling the balance between hydrolysis and transglycosylation and driving the elongation of β(1–3) glucan chains in the yeast cell wall.

Dandruff Is Associated with Disequilibrium in the Proportion of the Major Bacterial and Fungal Populations Colonizing the Scalp

The bacterial and fungal communities associated with dandruff were investigated using culture-independent methodologies in the French subjects. The major bacterial and fungal species inhabiting the scalp subject’s were identified by cloning and sequencing of the conserved ribosomal unit regions (16S for bacterial and 28S-ITS for fungal) and were further quantified by quantitative PCR. The two main bacterial species found on the scalp surface were Propionibacterium acnes and Staphylococcus epidermidis, while Malassezia restricta was the main fungal inhabitant. Dandruff was correlated with a higher incidence of M. restricta and S. epidermidis and a lower incidence of P. acnes compared to the control population (p<0.05). These results suggested for the first time using molecular methods, that dandruff is linked to the balance between bacteria and fungi of the host scalp surface.

β(1-3)Glucanosyltransferase Gel4p Is Essential for Aspergillus fumigatus

ABSTRACT The β(1-3)glucanosyltransferase GEL family of Aspergillus fumigatus contains 7 genes, among which only 3 are expressed during mycelial growth. The role of the GEL4 gene was investigated in this study. Like the other Gelps, it encodes a glycosylphosphatidylinositol (GPI)-anchored protein. In contrast to the other β(1-3)glucanosyltransferases analyzed to date, it is essential for this fungal species.