Identification and characterization of allergen components of the opportunistic yeast Malassezia furfur

Abstract: The yeast Malassezia furfur (Pityrosporum orbiculare) is a member of the human norrnal skin microflora. It has been suggested that this yeast plays an important role in the pathogenesis of atopic dermatitis (AD). In 40-65% of patients with AD anti-M. furfur activity has been shown with skin tests and/or specific IgE serology. The aim of this thesis was to identify and characterize the IgE binding components of this yeast. Monoclonal antibodies (MoAbs) against M. furfur were generated and two IgE-binding proteins of the yeast with the molecular mass of 36-kDa and 67-kDa were identified. Different extracts of M. furfur were compared by using ELISA, inhibition ELISA, SDS-PAGE and immunoblotting. The SDS-PAGE and immunoblotting results showed a great variation in the protein content of different extracts. Prolongation of culture periods for more than 4 days led to loss of most of the allergenic proteins while the carbohydrate content remained fairly constant. The high molecular mass glycoproteins or polysaccharides that are presumably involved in most of the IgE-binding capacity in extracts from old cultures are also present in comparable concentrations in preparations from 2-4 days of culture. Flow cytometry analysis of the M. furfur yeast cells cultured for 2 to 22 days revealed that the 36-kDa and 67-kDa allergens are expressed on the cell surface, with significantly decreased expression after more than 4 days of culture. These results indicate that preparations obtained from the exponential phase of yeast cultures (2-4 days old) should preferably be used in studies of the IgE response to M. furfur. The subcellular localization of the 36-kDa and 67-kDa allergens of M. furfur, M. sympodialis, M. pachydermatis, and various other yeasts was determined with confocal laser scanning microscopy and flow cytometry analysis. The members of Malassezia genus expressed the two allergens on the cell surface to a similar extent, whereas these proteins were virtually undetectable in the Candida genus and Saccharomyces cerevisiae. The sequence of the complete cDNA encoding the 36-kDa allergen of M. furfur was determined. The encoded protein is the first to be sequenced from M. furfur and it is a novel protein since no similarity was found with reported sequences in the data bases. The complete cDNA contains 1176 base pairs and the open reading frame is 1050 base pairs, with a deduced amino acid sequence of 350 residues. The leader sequence consists of 22 amino acids determining a mature protein of about 36 kDa. The recombinant Mal fl has been expressed in both prokaryotic (Escherichia coli) and eukaryotic (baculovirus infected insect cells) expression systems. The purified rMal fl from the two expression systems is able to react with IgE antibodies from atopic patients. Both the E. coli and baculovirus produced proteins can specifically inhibit IgE binding to a 36-kDa protein band in immunoblotting. This suggests that the recombinant proteins contain the majority, if not all the IgE binding epitopes of Mal fl . It is concluded that MoAbs and recombinant allergens are useful tools in studies on standardization and characterization of M. furfur allergen components. Recombinant allergens have the potential to improve diagnosis and treatment of allergic diseases.