Sophie Paris

Surface hydrophobin prevents immune recognition of airborne fungal spores

The air we breathe is filled with thousands of fungal spores (conidia) per cubic metre, which in certain composting environments can easily exceed 109 per cubic metre. They originate from more than a hundred fungal species belonging mainly to the genera Cladosporium, Penicillium, Alternaria and Aspergillus. Although these conidia contain many antigens and allergens, it is not known why airborne fungal microflora do not activate the host innate immune cells continuously and do not induce detrimental inflammatory responses following their inhalation. Here we show that the surface layer on the dormant conidia masks their recognition by the immune system and hence prevents immune response. To explore this, we used several fungal members of the airborne microflora, including the human opportunistic fungal pathogen Aspergillus fumigatus, in in vitro assays with dendritic cells and alveolar macrophages and in in vivo murine experiments. In A. fumigatus, this surface ‘rodlet layer’ is composed of hydrophobic RodA protein covalently bound to the conidial cell wall through glycosylphosphatidylinositol-remnants. RodA extracted from conidia of A. fumigatus was immunologically inert and did not induce dendritic cell or alveolar macrophage maturation and activation, and failed to activate helper T-cell immune responses in vivo. The removal of this surface ‘rodlet/hydrophobin layer’ either chemically (using hydrofluoric acid), genetically (ΔrodA mutant) or biologically (germination) resulted in conidial morphotypes inducing immune activation. All these observations show that the hydrophobic rodlet layer on the conidial cell surface immunologically silences airborne moulds.

Cloning and disruption of the gene encoding an extracellular metalloprotease of Aspergillus fumigatus

Aspergillus fumigatus secretes a serine alkaline protease (ALP) and a metalloprotease (MEP) when the fungus is cultivated in the presence of collagen as sole nitrogen and carbon source. The gene encoding ALP was isolated and characterized previously. We report here the cloning and the sequencing of the gene encoding MEP. Genomic and cDNA clones were isolated from A. fumigatus libraries using synthetic oligonucleotides as probes. Stretches of the deduced amino acid sequence were found to be in agreement with the N‐terminal amino acid sequence of MEP and with internal peptide sequences. The amino acid sequence of the enzyme contains a putative active‐site sequence HEYTH homologous to the active site of other bacterial and eukaryotic zinc metalloproteases. Sequence analysis reveals that MEP has a pre‐proregion consisting of 245 amino acid residues preceding the 388 amino acid residues of the mature region (molecular mass of 42 kDa). An alp mep mutant, deficient in proteolytic activity at neutral pH in vitro, was constructed and tested for pathogenicity in a murine model. No difference in pathogenicity was observed between the wild‐type strain and the alp mep double mutant, suggesting that ALP and MEP are not essential for the invasion of the lung tissues by A. fumigatus.

Nutritional studies on Paracoccidioides brasiliensis: the role of organic sulfur in dimorphism

The nutritional requirements of the mycelial and yeast-like phases of the dimorphic fungus Paracoccidioides brasiliensis, a human pathogen, were investigated. For all nine isolates tested, mycelial cells were prototrophic, whereas yeast-like cells required a sulfur-containing amino acid for growth. Moreover, changing the source of nitrogen greatly affected the morphology of the yeast-like cells.

Cyclic adenosine 3',5' monophosphate (cAMP) and dimorphism in the pathogenic fungus Paracoccidioides brasiliensis

Exogenous cAMP or its analogs inhibit the mycelium transformation of yeast and induce bulging of the apex of mycelia. But intracellular cAMP levels of yeast and mycelial cells are not significantly different.

Study of the virulence of some mutants of Beauveria brongniartii (= Beauveria tenella)

Abstract Ten lipase-negative mutants from a lipase-positive virulent strain of Beauveria brongniartii were found to be avirulent, similar to lipase-negative strains isolated from nature. Two morphological mutants and four auxotrophic mutants which were lipase positive were also avirulent. Hypotheses to explain these results are presented.

Physiological characteristics and virulence of auxotrophic and morphological mutants of Beauveria Brongniartii (sacc.) petch (= Beauveria tenella)

Various mutants have been obtained of B. brongniartii, a hyphomycete pathogenic for insects. Both one pigment less and two blastospore negative mutants retained their initial virulence. Thus neither the characteristic parasitic blastospore morphology nor the synthesis of the pigment appeared to be required for pathogenesis. Analysis of avirulent mutants indicated that chitinolytic activity may be essential for fungal penetration of the cuticle.

EXOANTIGENS OF ASPERGILLUS FUMIGATUS: SERODIAGNOSIS AND VIRULENCE

Aspergillus fumigatus commonly grows in damp environments such as soil, decaying vegetation and organic debris from which it releases a high number of spores into the atmosphere (Raper and Fennell, 1965). The number of A. fumigatus conidia present in the air usually varies from 0.1 to 10 spores/m3, but may reach 106/m3 under certain environmental conditions (Mullins et al., 1976; Bodey and Vartivarian, 1989; Gumowski et al., 1992). Due to their small size (2–3 μm) and presence in the air, A. fumigatus conidia are inhaled by all individuals and transit through the upper respiratory tract, bronchia and bronchiola to end in the alveoli (Cole and Samson, 1984). The pulmonary epithelium is the largest epithelial surface exposed to the external environment (40–80 m2/individual). It possesses several lines of defence capable of expelling A. fumigatus from the lung (Seaton et al., 1989). Impairment of these defence mechanisms can lead to the establishment of the fungus in the lung. This impairment can be of genetical (cystic fibrosis, chronic granulomatous disease) or clinical origin (tuberculosis, chemo- and corticotherapy) (Cohen et al., 1981; Wagner, 1984; Schonheyder et al, 1988; Bodey and Vartivarian, 1989; Cohen, 1991). There are three clinical manifestations of A. fumigatus infections: 1) allergie bronchopulmonary aspergillosis (ABPA) resulting from repeated inhalation of conidia with limited fungal growth; 2) aspergilloma where colonisation of a pre-existing pulmonary cavity forms a fungal ball; 3) invasive pulmonary aspergillosis where A. fumigatus invades lung parenchyma and disseminates to other organs.

Heterocaryons chez Beauveria tenella.

Several methods were used in an attempt to obtain heterokaryons. They were only formed by fusion with polyethylene glycol of two nutritionally complementing protoplasts. Their nuclear ratio was <1 and they were not stable.ResumeDifférentes méthodes pour obtenir des hétérocaryons entre mutants auxotrophes ont été utilisées. Seule la méthode de fusion par le polyéthylène glycol a donné des hétérocayons; ces hétérocaryons ont un rapport nucléaire inférieur à 1 et sont instables.

Cytochemical study of the yeast and mycelial cell walls ofParacoccidioides brasiliensis

Abstract Cell wall structure and macromolecular organization of the various growth forms of Paracoccidioides brasiliensis were investigated using chemical, enzymatic, and cytochemical methods. The wall of yeast-like cells was not sensitive to periodic acid and β(1 → 3) glucanase treatments, but bound calcofluor suggesting that it is composed of an α(1 → 3) glucan and of chitin. The fibrillar outer layer of bud cell initials and of abscision areas, were characterized by the presence of a strongly periodic acid- and concanavalin A-reactive substance that was sensitive to the lytic action of protease, presumably composed of mannan and proteins. The outermost layer of the mycelium was sensitive to protease, and periodic acid and peanut agglutinin positive indicating that it was composed of galactomannan and protein. The inner layer is mainly composed of a β(1 → 3), (1 → 6) glucan (sensitive to snail enzyme but not to an exo β(1 → 3) glucanase), and chitin. Septa were brightly fluorescent with calcofluor. Our results are compared with models previously proposed by other authors.

Allergens Of Alternaria And Cladosporium

Cladosporium and Alternaria are among the most widespread airborne fungi. The conidia of these fungi are present indoors and outdoors all year around with a peak during mid and late summer. Eventhough the spore counts vary according to the climatic and geographic conditions, the mean concentration of spores during the peak season is usually higher for Cladosporium than Alternaria (5,000 to 250,000 vs 200 to 5,000 spores per m3 of air respectively) (Fig. 1). However the volume of one spore of Alternaria is typically 20 to 40 times greater than that of Cladosporium. Consequently, the difference in the realtive abundance of these two genera becomes less significant if the volume contribution is taken into account (Mallea and Charpin, 1980; Lacey, 1981; Larsen, 1981; A1 Doory, 1984; Beaumont et al., 1985).