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Summary Common indoor and outdoor environmental fungi such as Aspergillus fumigatus produce asexual spores containing a collection of proteins that can bind IgE antibodies and trigger allergic reactions. We characterized the impact of sporulation temperature on the IgE-binding capacity (allergenicity) of A. fumigatus and explored the links between variable allergenicity and temperature-dependant expression of genes encoding these allergenic proteins. A 12-fold increase in A. fumigatus allergenicity per spore was observed when sporulation temperatures were decreased from 32°C to 17°C. Per spore protein mass and Asp f 1 allergen mass also followed this trend. Functional gene expression analysis of A. fumigatus sporulating cultures by real-time reversetranscription PCR and gene expression microarrays revealed that a greater number of genes encoding known, major allergens are more highly expressed at lower sporulation temperatures. The results of this study indicate that environmental conditions at growth significantly influence the allergenicity of this common mold through the differential production of allergenic proteins, and highlight the importance of in vivo or in vitro allergenicity measurements for understanding environmental exposure to airborne allergenic fungi.

(1→3)-β-D-glucans are structural cell wall components of fungi, plants, and some bacteria and have been linked with human respiratory symptoms following aerosol exposure. A clear interpretation of the health impact of (1→3)-β-D-glucans is limited by the high cost and uncertainties associated with current glucan quantitation methods. The objective of this research is to develop DNA aptamers for the measurement of (1→3)-β-D-glucans. Aptamers are synthetic DNA functional binding molecules that fold into unique conformations, allowing them to bind specifically to their target. Through the in-vitro selection process SELEX, we have produced aptamers that are able to bind with sub-micromolar affinity to curdlan, a linear unbranched form of (1→3)-β-D-glucans. These aptamers display high selectivity to curdlan and do not bind to non-(1→3)-β-D polysaccharides, suggesting specificity for the β-(1→3)-glycosidic linkage. The aptamers produced here will enable the production of more cost effective, less ambiguous assays for the environmental measurement of (1→3)-β-D-glucans.

Allergies are caused by the binding of IgE antibodies onto specific sites on allergens. However, in the assessment of exposure to airborne allergens, current techniques such as whole spore counts fail to account for the presence of these allergenic epitopes that trigger allergic reactions. The objective of the research is to develop a DNA aptamer for the Asp f 1 allergen of the pathogenic fungus Aspergillus fumigatus, using an IgE-binding epitope of the allergen as the target for aptamer selection. Through in vitro SELEX, an aptamer has been produced that binds with nanomolar affinity to the Asp f 1 IgE-epitope. The aptamer is also able to recognize the native Asp f 1 allergen, and does not bind to allergenic proteins from non-target mold species such as Alternaria alternata. Production of this aptamer provides proof-of-principle that allergen measurement methods can be developed to indicate the potent fraction, or allergenicity, of allergens.

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