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I - Pathobiology of Aspergillus fumigatus
In spite of the dramatic increase in the incidence of invasive aspergillosis, the pathogenicity of A. fumigatus is still poorly understood. The two strategies presently undertaken in our laboratory to tackle this problem, result from previous molecular studies suggesting that virulence of A. fumigatus is polygenic. The first approach is the study of the interactions between the alveolar macrophage (AM) and the conidia of A. fumigatus landing in the alveolus after inhalation (Granet, Philippe). The phagocytic events have been characterized. Actin plays an essential role in the early step of the internalization of conidia by AM ; the conidia are then quickly adressed to the phagolysosome. Intramacrophagic killing of the conidia is extremely slow and is highly reduced by glucocorticoïds. The role of different macrophage effectors in the killing of conidia (reactive oxidants, nitric oxide, lysosomial enzymes) is presently investigated in 3 different models: resistant immunocompetent mice, susceptible immunocompromised or transgenic mice and vaccinated immunocompromised mice. The second approach is a transcriptome/proteome analysis of genes and proteins expressed during the early stages of the infection (Buitrago, Mouyna, Beauvais). These studies, now possible with the ongoing sequencing of the genome of A. fumigatus, are undertaken in collaboration with the GMP Laboratory and use wild-type and mutant strains constructed in our laboratory, with different levels of virulence.
II - Structural characterization and biosynthesis of the cell wall of the mycelium and conidium of A. fumigatus
Major polysaccharides of the cell wall of A. fumigatus are a and ß(1-3) glucans. Biosynthesis of ß(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 FKSp, the putative catalytic subunit using UDP-glucose as the substrate. Three genes AGS involved in the biosynthesis of a (1-3) glucans have been identified and the phenotype of the AGS mutants is presently under study (Beauvais).
In the periplasmic space, neosynthesized ß1-3 glucans are modified and associated to the other cell wall polysaccharides to produce the rigid three-dimensional network characteristic of the cell wall. Chemical characterization of the A. fumigatus cell wall has shown that the central core of the cell wall is a branched ß1-3/1-6 glucan to which are attached chitin and galactomannan (Fontaine). Such organization requires the presence of active glycosyltransferases in the periplasmic space. During the search for such activities, it was shown for the first time that proteins 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 characterization of the GPI anchor and its biosynthesis in A. fumigatus has been studied (Fontaine). In addition, the role of the dozen of GPI-anchored proteins, common to both filamentous fungi and yeast is presently investigated using a combination of molecular biology and carbohydrate chemistry techniques (Mouyna, Chabane, Morelle, Bernard, Fontaine).
Similar approaches are undertaken to study the conidium cell wall since it plays an essential role in the survival of the infective conidium in the lung of the immunocompetent patients (Paris). |
