Cloning and disruption of the antigenic catalase gene of Aspergillus fumigatus

Abstract

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...

Aspergillus fumigatus catalases: cloning of an Aspergillus nidulans catalase B homologue and evidence for at least three catalases

“…These results demonstrate the presence of a third catalase protein, CatC, produced by A. fumigatus, which has a high level of identity to CatB. The gene for CatB has been cloned and both the gene and protein were analysed in detail in both this report and in two previous reports where it is termed Cat1 [3,4]. In addition, a mutant strain, vcatB, has been generated in which the catB gene has been disrupted [4].…”

“…The DNA database accession number of the A. fumigatus catB gene described here is Y07763. This gene has also been submitted as U87850 by Wysong et al [18] and U97574 by Calera et al [4].…”

“…A. fumigatus strain G10 is a spontaneous nitrate reductase mutant of a clinical isolate CBS144-89. Strain G10 has been used to generate the mutant, vcatB, which has been disrupted in one of the catalase genes, catB [4].…”

“…The expression of catalases was investigated in 1day cultures of three strains: AF10 is a clinical isolate; a mutant (vcatB) was obtained [4] in which the catB gene had been disrupted and G10 is the parental strain of the mutant. The water soluble fractions were extracted and analysed using non-denaturing PAGE.…”

“…It has been previously reported that A. fumigatus produces two catalases and one catalaseperoxidase di¡erentiated on the basis of their electrophoretic mobility [2]. In addition, one puri¢ed A. fumigatus catalase (Cat1) has been shown to be resistant to reducing and denaturing agents, to high temperatures and is a tetramer comprised of 90-kDa subunits [3,4]. Antibodies to this A. fumigatus catalase are frequently detected in aspergilloma patients [3,5].…”