Studies on the entomopathogenic fungus Beauveria bassiana: Molecular and immunological characterization of allergens.

摘要

Beauveria bassiana is an entomopathogenic fungus currently under development as a biological control agent against a wide range of arthropod pests. Although B. bassiana has been reported to be non-toxic to vertebrates, its potential allergenicity has not been studied. Fungal allergens constitute a significant proportion of the airborne allergens that affect up to 25% of the population of the industrialized world. This dissertation examines the ability of B. bassiana to elicit allergic reactions, and describes the immunological and molecular characterization of IgE binding proteins present in this fungus.;Immunoblot analyses of B. bassiana proteins probed with pooled and individual human sera revealed IgE reactive antigens, ranging from 12 to >95 kDa. Variation was noted when blots were probed using individual sera, however a 35 kDa protein was the most frequently reactive B. bassiana antigen. Immunoblot inhibitions experiments identified the presence of shared epitopes between B. bassiana and the extracts of several common allergenic fungi (cross-reactivity). IgE binding of the 35 kDa protein was not inhibited by any of the fungal extract tested, indicating the possible presence of a B. bassiana specific antigen. Intradermal skin testing confirmed the in vitro results, demonstrating allergenic reactions in a number of individuals, including those who have had occupational exposure to B. bassiana.;Screening of a B. bassiana cDNA library revealed a number of proteins with sequence similarity to major fungal allergens. Full length clones of the B. bassiana genes were obtained by 3' and 5' RACE PCR, and designated as; bbenol, bbf2, bbald, and bbhex. All four proteins were expressed in E. coli. BbEnol, designated an enolase by sequence similarity, was compared to 20 other fungal enolases including five known to be allergenic and cross-reactive. Phylogenic comparison showed allergenic (and cross-reactive) enolases are not limited to closely related taxa, but are equally distributed throughout the phylogram. Immunoblot analysis of the four B. bassiana proteins revealed BbEnoI and BbAld to be reactive to sera IgEs, and therefore represent the first allergens to be identified from the entomopathogenic fungus Beauveria bassiana.