cDNA cloning, expression and characterization of an allergenic L3 ribosomal protein of Aspergillus fumigatus

Abstract

Aspergillus fumigatus (Afu) is an important fungal pathogen causing allergic and invasive respiratory disorders. A plethora of multi-functional allergens/antigens secreted by Afu have been implicated in pathogenesis. The present study was undertaken to identify and characterize novel Afu allergen/antigen by cDNA library approach. cDNA library of Afu was immunoscreened with pooled sera of allergic bronchopulmonary aspergillosis (ABPA) patients. The cDNA clone, TS1, reacting significantly with specific IgG antibodies, was selected. cDNA was subcloned and expressed in Escherichia coli. Sequencing of the cDNA revealed an open reading frame (ORF) of 1179 bases coding for a protein with an approximate molecular weight of 44 kDa. Immunoreactivity of the recombinant TS1 protein (rTS1) was evaluated by ELISA and Western blot analysis using pooled sera of ABPA patients. The rTS1 exhibited binding to specific IgG and IgE antibodies present in sera of ABPA patients. The deduced amino acid sequence showed homology to 60S ribosomal protein L3 (RpL3) of Aspergillus nidulans, Saccharomyces cerevisiae and Homo sapiens. The RpL3 of S. cerevesiae, tcm1, to which TS1 sequence shows significant homology (72% identity), is known to be responsible for conferring resistance against trichodermin (antibiotic, inhibiting protein synthesis). The present study has led to identification, cloning and expression of a 44-kDa novel allergen/antigen of Afu with sequence homology to L3 ribosomal protein with a probable role in resistance of Afu to antifungal drugs. Sixty-four per cent sequence identity of Afu RpL3 with human RpL3 and common regions in their predicted epitopes suggest a possibility of involvement of Afu RpL3 in autoimmune reactions due to molecular mimicry.