Unit: Aspergillus

Director: Jean-Paul Latgé

Aspergillus fumigatus is a thermophilic, saprophytic filamentous fungus that sporulates abundantly in nature. Pulmonary diseases consecutive to the inhalation of airborne conidia of A. fumigatus are often fatal among immunocompromised patients. Indeed, A. fumigatus has become today the most important fungal aerial pathogen in developed countries. The study of aspergillosis is the main focus of the Aspergillus Unit The first research theme of the laboratory is medically orientated and is focused on the study of invasive aspergillosis (IA). The other research area is more fundamental and concerns the study of the fungal cell wall biosynthesis that is a key morphogenetic event in the fungal life. In addition, such studies should identify targets for the development of new antifungal drugs that are urgently needed for the treatment of fungal diseases.

I - Pathobiology of Aspergillus infections

To understand the early stages of the establishment of A. fumigatus in the lung parenchyma, the interactions between the alveolar macrophage (AM) that is the main phagocytic cells of the lung environment and the conidia of A. fumigatus have been studied. Killing of the conidia in the immunocompetent host is due to reactive oxidant intermediates associated to the acidification of the phagolysosome. Activation of the MAP kinases ERK and p38 and translocation of NFkB were associated to the phagocytosis of conidia by AM. Glucocorticoïds are responsible for a reduction of reactive oxidant species and an inhibition of the phosphorylation of ERK and p38. These cellular events that resulted in an intracellular germination of conidia in the AM, explain a posteriori why glucocorticoïds trigger the development of IA among immunocompromised patients (Granet).

In accordance with the phagocytosis data, analysis of virulence factors of A. fumigatus has been now focused on the study of mutants affected in sensitivity to oxidative stress. However, mutants of A. fumigatus deficient in catalases or in specific transcription factors such as Skn7 or signal transduction cascades such as Hog1, that are sensitive to reactive oxidants in vitro are as pathogenic as parental strains in an experimental murine aspergillosis model. These data.suggest that in addition of reactive oxidants, other toxic molecules produced by the phagocytes are involved in the killing of A. fumigatus in vivo. To identify genes concomitanly expressed during infection, a transcriptome analysis has been initiated with the genopole of Toulouse and an european consortium. First analysis have concerned the early steps of the germination (Lamarre).

II - Diagnosis of aspergillosis

New serological diagnostic methods are currently investigated since a better therapy of invasive aspergillosis patients is depending on progresses in the diagnosis of this disease. Our unit has played a major role in the chemical and immunological characterization of the galactomannan of A. fumigatus that is at the origin of the development of the only commercial kit for the diagnosis of IA. This kit is a sandwich ELISA based on a monoclonal antibody detecting circulating beta1-5 galactofuran. Recent studies have shown that the quantification of anti-A. fumigatus antibodies using recombinant protein antigens is useful to identify patients at risk for IA (Sarfati).

III - Structure and biosynthesis of the cell wall of A. fumigatus

Major polysaccharides of the cell wall of A. fumigatus are alpha and beta(1-3) glucans and chitin. Biosynthesis of beta(1-3) glucans is under the control of FKS1, a unique essential membrane protein. In contrast, chitin and alpha(1,3) glucan are synthesized by families of genes that have different functions. Synthesis of chitin is under the control of a chitin synthase gene family composed of 8 members. Disruption of only 2 members of this family lead to a growth phenotype. Three AGS genes involved in the biosynthesis of alpha(1-3) glucans have been identified and only 1 of the AGS mutants has a reduced content of alpha(1-3) glucans. These results suggest that compensatory mechanisms exist in the construction of the cell wall and these mechanisms are studied through a transcriptome approach (Beauvais).

Remodeling of the cell wall polysaccharides is due to two groups of enzymes. The first group is composed of glycosylhydrolases able to dissolve the conidial cel wall during germination (Debeaupuis). The second group is constituted of glycosyltransferases that branch the neosynthesized beta(1-3) glucans and link it to the other cell wall polysaccharides (chitin, galactomannan and beta1-3, 1-4 glucan). During the search for such activities, it was shown for the first time that glycosyltransferases bound to the membrane by a glycosylphosphatidyl inositol (GPI) anchor play a major role in the biosynthesis of the yeast and mold cell wall. Chemical structure of the GPI anchor and its biosynthesis in A. fumigatus have been elucidated (Fontaine). Five families of GPI-anchored proteins, common to both filamentous fungi and yeast have been identified following a proteome and comparative genomic analysis of GPI-anchored proteins of S. cerevisiae and A. fumigatus and are now studied using a combination of biochemistry and molecular biology techniques (Mouyna).

Keywords: Aspergillus, lung, aspergillosis, cell wall, glucan, alveolar macrophage

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