Aspergillus fumigatus possesses two catalases (described as fast and slow on the basis of their electrophoretic mobility). The slow catalase has been recognized as a diagnostic antigen for aspergillosis in immunocompetent patients. The antigenic catalase has been purified. The enzyme is a tetrameric protein composed of 90-kDa subunits. The corresponding cat1 gene was cloned, and sequencing data show that the cat1 gene codes for a 728-amino-acid polypeptide. A recombinant protein expressed in Pichia pastoris is enzymatically active and has biochemical and antigenic properties that are similar to those of the wild-type catalase. Molecular experiments reveal that CAT1 contains a signal peptide and a propeptide of 15 and 12 amino acid residues, respectively. cat1-disrupted mutants that were unable to produce the slow catalase were as sensitive to H 2 O 2 and polymorphonuclear cells as the wild-type strain. In addition, there was no difference in pathogenicity between the cat1 mutant and its parental cat1 ؉ strain in a murine model of aspergillosis. MATERIALS AND METHODS Strains and culture conditions. A. fumigatus AFN (26) and CBS144-89 (Centraalbureau voor Schimmelcultures, Baarn, The Netherlands) are clinical isolates. Strain G10, a spontaneous nitrate reductase mutant of strain CBS144-89 (30), was chosen as the recipient strain for transformations. Escherichia coli LE392 and Y1090 were used for propagation of bacteriophages EMBL3 and gt11, respectively. All plasmid subcloning experiments were performed in E. coli DH5␣. Three media were used to grow A. fumigatus at 25°C on shaken flasks or on a fermentor: Sabouraud medium (2% glucose, 1% Mycopeptone Biokar; Prolabo, Beauvais, France), 1% yeast extract medium (Difco, Detroit, Mich.), and Bacto Czapek-Dox broth (Difco). Electrophoresis and immunoblotting. Nondenaturing electrophoresis and SDS-PAGE were performed as previously described (26) with the discontinuous buffer system (23). Immunoblotting was performed by the method of Towbin et al. (42). The glycosylation of CAT1 was detected on Western blots by the concanavalin A (ConA)-peroxidase-conjugated technique of Fontaine et al. (6). Catalase activity. Catalase activity was observed in phosphate-buffered saline (PBS) containing 0.1 M H 2 O 2 by the release of O 2 bubbles. For the detection of catalase activity on nondenaturing gels, water-soluble extracts obtained by mycelial disruption as previously described (12) were subjected to nondenaturing PAGE, and the ferricyanide-negative stain of Woodbury et al. (47) was used to locate bands containing catalase activity as described by Wayne and Diaz (46). Purification of CAT1. The water-soluble fraction of A. fumigatus (strain CBS144-89, grown in Sabouraud medium), obtained by the method of Hearn et al. (13), was used as the starting material. A Mono Q HR 5/5 column (Pharmacia, Uppsala, Sweden) was used for the anion-exchange purification step. Samples were loaded in 10 mM Tris-HCl (pH 7.5) and eluted with a linear NaCl gradient (0 to 350 mM in 30 min). Gel filtration c...

We have identified, purified, and characterized structurally and functionally a 90-kDa immunodominant antigen associated with the water-soluble fraction of Aspergillus fumigatus. This antigen is recognized by 90.3% of serum samples from patients with aspergilloma and should be considered either by itself or better in combination with other purified antigens as a candidate for developing a standardized immunoassay for the detection of aspergilloma. p90 is a glycoprotein containing at least two N-linked sugar chains of 2 and 5 kDa, respectively, which are not necessary for its reactivity with aspergilloma serum samples. Using specific anti-p90 rabbit serum, we have demonstrated that under native conditions, p90 exists in oligomeric form and has associated catalase activity. This activity is resistant to extreme temperatures (>60؇C), reducing agents (40 mM dithiothreitol), high concentrations of denaturing agents such as 8 M urea and 8% sodium dodecyl sulfate, and treatments with ethanol-chloroform-water (5:3:10 [vol/vol]) mixtures.

For the first time, an immunodominant Aspergillus nidulans antigen (ASPND1) consistently reactive with serum samples from aspergilloma patients has been purified and characterized, and its coding gene (aspnd1) has been cloned and sequenced. ASPND1 is a glycoprotein with four N-glycosidically-bound sugar chains (around 2.1 kDa each) which are not necessary for reactivity with immune human sera. The polypeptide part is synthesized as a 277-amino-acid precursor of 30.6 kDa that after cleavage of a putative signal peptide of 16 amino acids, affords a mature protein of 261 amino acids with a molecular mass of 29 kDa and a pI of 4.24 (as deduced from the sequence). The ASPND1 protein is 53.1% identical to the AspfII allergen from Aspergillus fumigatus and 48% identical to an unpublished Candida albicans antigen. All of the cysteine residues and most of the glycosylation sites are perfectly conserved in the three proteins, suggesting a similar but yet unknown function. Analysis of the primary structure of the ASPND1 coding gene (aspnd1) has allowed the establishment of a clear relationship between several previously reported A. fumigatus and A. nidulans immunodominant antigens. The increasing incidence of Aspergillus-related diseases (43) has prompted many investigators to search for fungal molecules relevant either for the immunodiagnosis of or for understanding the pathology of the different forms of aspergillosis (ranging from pulmonary aspergilloma to invasive aspergillosis, passing through allergic situations). The role of several putative virulence factors has been investigated without too much success (for a review, see reference 3). In contrast, several antigens of demonstrated value as immunodiagnostic reagents for different Aspergillus infections have been purified, and some of their coding genes have been cloned. The AspfI ribotoxin (27), the AspfII allergen (2), and heat shock protein 1 (HSP-1) (16) from Aspergillus fumigatus have been obtained as recombinant antigens. The CAT1 (the subunit of a catalase) (22) and superoxide dismutase (13) antigens have been purified to homogeneity from A. fumigatus water-soluble extracts. All of these antigens have been shown to be consistently reactive with sera from patients with different forms of aspergillosis but not with sera from control or healthy individuals (2, 10, 24, 26). Certain other purified molecules, such as the 58-kDa (6) and gp55 (40) antigens, or semipurified fractions, such as the CS2 complex (30) or the cytosolic fraction complex (CFC) (24) from A. fumigatus, have been claimed to be useful reagents for immunodiagnosis. In this paper, we attempt to simplify the entangled antigenic array of A. fumigatus antigens by using data obtained from the purification and immunochemical characterization of a previously reported Aspergillus nidulans antigen (4) (now designated ASPND1), cloning of its coding gene, and analysis of its primary structure. The conclusion of this study is that several previously reported antigens or antigenic Aspergillus preparations conta...

The elderly account for a disproportionate share of all tuberculosis cases, and the population ageing may not fully explain this phenomenon. We have performed in vitro infection experiments to investigate whether there is an immunological basis for the apparent susceptibility of elders to tuberculosis. In our infection model, Mycobacterium tuberculosis induces a higher production of interleukin (IL)-6 and reactive oxygen species in macrophages from elders than from younger adults. This response did not prevent, however, an increased multiplication of M. tuberculosis in macrophages from elders as compared with the growth observed within cells from adults. By performing a factorial experiment, we have found that IFN-γ, but not IL-1β, IL-6 or TNF-α, stimulate the macrophages to restrict the multiplication of the bacterium in macrophages from elders. Although monocytes from elders seem to be in a higher level of activation, we present evidences that protein tyrosine phosphorylation response induced by M. tuberculosis is stronger in monocytes from adults than from elders. Using a protein array that detects 71 tyrosine phosphorylated kinases, we identified Pyk2 as the only kinase that displayed a difference of intensity larger than 50 % in adults than in elders. Furthermore, monocytes from elders that were incubated in the presence of tyrosine kinase inhibitors (genistein and PP2) allowed a higher level of bacterial multiplication. These observations may help to explain the susceptibility of elders to tuberculosis. An unexpected result was that both genistein and its negative control, daidzein, abundant soy isoflavones, promoted intracellular mycobacterial growth.

Cytosolic fractions of mycelial extracts from Aspergillus nidulans, A. flaws, and three different isolates of A. fumigatus, grown to stationary phase in Czapek-Dox-AOAC medium, were tested by immunoblotting for the presence of antigens reactive to 80 serum samples from aspergilloma patients. Fifty control serum samples were used to determine the specificity of the reactions. In the A. fbmigatus cytosolic fraction a group of four main antigenic bands (p90, p60, p40 and p37) was consistently recognized (in total or partial form) by 90% of the serum samples from the aspergilloma patients. This group of antigens was designated as the 'cytosolic fraction complex' (CFC). As confirmed by two-dimensional electrophoresis followed by immunoblotting with aspergilloma serum samples, each of the four antigenic bands is formed of several isoforms of acidic glycopeptides with slightly different pls. All the isoforms are at least N-glycosylated, as demonstrated by endoglycosidase H removal of a considerable amount of sugar residues. The relationship of these antigens with certain other A. fumigatus antigens previously reported in the literature, and their potential use in the immunodiagnosis of aspergilloma, are discussed.

Aspergillus nidulans catalase B (CatB) was purified to homogeneity and characterized as a hydroperoxidase which resembles typical catalases in some physicochemical characteristics: (1) it has an apparent molecular weight of 360 000 and is composed of four glycosylated subunits, (2) it has hydrophobic properties as revealed by extractability in ethanol/chloroform and binding to phenyl-Superose, and (3) it has an acidic isoelectric point at pH 3.5. Also CatB exhibits some distinctive properties, e.g. it is not inhibited by the presence of 2% sodium dodecyl sulfate, 9 M urea or reducing agents. Furthermore, even though CatB does not exhibit any residual peroxidase activity, it is able to retain up to 38% of its initial catalase activity after incubation with the typical catalase inhibitor 3-amino-1,2,4-triazole. ß

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The influence of culture medium and time and temperature of growth on the appearance of Aspergillus nidulans antigens was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by silver staining or Western blot (immunoblot), of the proteins present in total cellular extracts or culture supernatants. Samples in the exponential, deceleration, and stationary growth phases were selected by