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  Director : Jean-Paul Latgé (jplatge@pasteur.fr)


  abstract

 

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 first research theme of the laboratory is medically orientated and is focused on the study of invasive aspergillosis. Special emphasis is directed towards the analysis of the host-pathogen interactions to understand the early stages of the establishment of A. fumigatus in the lung parenchyma. On the applied side, new serological diagnostic methods are currently investigated since a better management of invasive aspergillosis patients is based on a better diagnosis of this mycosis. 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 could lead to the identification of targets for the development of new antifungal drugs that are urgently needed for the treatment of fungal diseases.



  report

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I - Pathobiology of Aspergillus fumigatus

The interactions between the alveolar macrophage that is the main phagocytic cells of the lung environment and the conidia of A. fumigatus have been studied (Granet, Philippe). The intracellular trafficking of the conidium towards the phagolysosome has been specifically investigated. Killing of the conidia in the immunocompetent host is due to reactive oxidant intermediates associated to the acidification of the phagolysosome. Glucocorticoids highly reduced the production of reactive oxidants, resulting in the intracellular germination of conidia that initiates the establishment of A. fumigatus in the lung . This finding explains a posteriori why glucocorticoids triggers the development of invasive aspergillosis among immunocompromised patients. However, mutants of A. fumigatus that are deficient in catalase, that are supposed to counteract the host reactive oxidant species, are as pathogenic as parental strains in an experimental murine aspergillosis model. These results and the analysis of other A. fumigatus mutants obtained by reverse genetics suggest that the virulence of A. fumigatus is polygenic (Paris). To approach this problem, and to identify genes concomittanlty expressed during infection, a transcriptome analysis has been started. Surch large scale investigation studies are now feasible since the sequence of the genome of A. fumigatus (11 mb, >11000 genes) is now available (Beauvais, Debeaupuis, Henry, Mouyna, Sarfati).

The serological diagnosis of aspergillosis is based on the immunochemical detection of several glycoproteins (produced as recombinant proteins) (Sarfati) and of galactofuran-bearing molecules. The galactomannan is a major antigen of A. fumigatus that is at the origin of the development of the only commercial kit for the diagnosis of invasive aspergillosis. This kit is a sandwich ELISA based on a monoclonal antibody detecting circulating b1-5 galactofuran. New A. fumigatus molecules bearing this epitope have been characterized: (i) glycoproteins with terminal galactofuranose at the non-reducing end of the mannan core and (ii) a lipophosphogalactomannan (Costachel, Fontaine Bernard).

II - Structural characterization and biosynthesis of the cell wall of A. fumigatus

Major polysaccharides of the cell wall of A. fumigatus are a and b(1-3) glucans and chitin. Biosynthesis of b (1-3) glucans is under the control of a membrane protein complex, the glucan synthase. This complex is composed of 2 essential members, Rho1p, a regulatory GTPase and FKS1p, the putative catalytic subunit using UDP-glucose as the substrate. Both genes are essential for A. fumigatus. Synthesis of chitin is under the control of a chitin synthase gene family composed of 7 members. Only disruption of 2 members of this family lead to phenotype with reduced growth and altered conidial permeability. Three AGS genes involved in the biosynthesis of a (1-3) glucans have been identified and the AGS mutants are characterized by a reduced growth and altered conidiation (Beauvais).

In the periplasmic space, neosynthesized b1-3 glucans are modified and associated to the other cell wall polysaccharides (chitin, galactomannan and b1-3, 1-4 glucan) to produce the rigid three-dimensional network characteristic of the cell wall. Such organization requires the presence of active transglycosidases in the periplasmic space. During the search for such activities, it was shown for the first time that glucanosyltransferases bound to the membrane by a glycosylphosphatidyl inositol (GPI) anchor play a major role in the biosynthesis of the yeast and mold cell wall (Mouyna). Chemical characterization of the GPI anchor and its biosynthesis in A. fumigatus has been also investigated (Fontaine). A dozen 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. Their role in the biosynthesis of the A. fumigatus cell wall is presently investigated using a combination of molecular biology, carbohydrate and protein chemistry techniques (Bernard, Chabane, Mouyna).

Figure 1: Electron microscopy and immunofluorescence views of conidia (in green) of A. fumigatus killed or germinating, in the alveolar macrophage after 24 h in vivo infection. Upper part : immunocompetent mice. The white and black arrows indicated dead conidia. Lower part: cortisone acetate immunocompromised mice. The conidia germinated (black arrow). The germ tubes (in red) were detected using an anti-A. fumigatus antibody revealed by a secondary antibody conjugated to Texas-red.

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



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  publications

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  personnel

  Office staff Researchers Scientific trainees Other personnel
  CORMIER Marinette (mcormier@pasteur.fr) BEAUVAIS Anne, IP (Scientist,abeauvai@pasteur.fr)

FONTAINE Thierry, IP (Scientist,tfontaine@pasteur.fr)

GRANET Oumaïma, IP (Scientist,ogranet@pasteur.fr)

MOUYNA Isabelle, IP (Scientist,imouyna@pasteur.fr)

PARIS Sophie, IP (Scientist,sparis@pasteur.fr)

BERNARD Muriel (PhD student,mbernard@pasteur.fr)

CHABANE Sandrine (Postdoctoral fellow,schabane@pasteur.fr)

COSTACHEL Corina (Postdoctoral fellow,ccostach@pasteur.fr)

PHILIPPE Bruno (PhD student,bphil@pasteur.fr)
DEBEAUPUIS Jean-Paul, INSERM (Research assistant, mailto:jpdbp@pasteur.fr)

HENRY Christine, IP (Research assistant,chenry@pasteur.fr)

SARFATI jacqueline, INSERM (Research assistant,jsarfati@pasteur.fr)

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