Identification of Immunoglobulin E-Binding Proteins of the Xerophilic Fungus Aspergillus penicillioides Crude Mycelial Mat Extract and Serological Reactivity Assessment in Subjects with Different Allergen Reactivity Profiles

Abstract

Background: Aspergillus penicillioides is a very common indoor xerophilic fungus and potential causative agent of respiratory conditions. Although people are constantly exposed to A. penicillioides, no proteins with allergenic potential have been described. Therefore, we aim to confirm allergic sensitization to A. penicillioides through reactivity in serological assays and detect immunoglobulin E (IgE)-binding proteins.

Methods: In an indirect ELISA, we compared the serological reactivity to A. penicillioides between subjects with specific IgE (sIgE) (group 1, n = 54) and no sIgE reactivity (group 2, n = 15) against commercial allergens. Correlations and principal component analysis were performed to identify associations between reactivity to commercial allergens and A. penicillioides. IgE-binding proteins in A. penicillioides were visualized using Western blotting (WB) in group 1. The IgE-binding proteins with the highest reactivity were analyzed by mass spectrometry and confirmed by transcript matching.

Results: There was no statistical significance (p = 0.1656) between the study groups in serological reactivity. Correlations between reactivity to A. penicillioides, dog epithelia, Aspergillus fumigatus, and Penicillium chrysogenum were observed. WB experiments showed 6 IgE-binding proteins with molecular weights ranging from 45 to 145 kDa. Proteins of 108, 83, and 56 kDa showed higher reactivity. Mass spectrometry analysis of these 3 proteins led to the putative identification of NADP-specific glutamate dehydrogenase and catalase B. This was confirmed with transcriptome analysis.

Conclusions: These results provide evidence of the presence of potential allergenic components in A. penicillioides. Further analysis of the putatively identified proteins should reveal their allergenic potential.

Keywords: Aspergillus penicillioides; Allergy; Immunoglobulin E-binding proteins; Serologic reactivity; Xerophilic fungi.

Figure 1

Indirect ELISA reactivity results (A₄₉₀) of human sera against the crude mycelial mat extract (MME) of A. penicillioides. The x-axis represents the serum sample groups (group 1, n = 54; group 2, n = 15). The y-axis represents the absorbance values for each individual. The red squares within each group represents the median value for each group. The red line represents the A₄₉₀ for the negative control.

Figure 2

Correlation circle from Factors 1 and 2 illustrating the correlation between allergens. Variability percentage for factor 1 (x-axis) is 46.48%, for factor 2 (y-axis) is 18.08%, for a cumulative variability of 64.46%. The x-axis represents the correlations between the variables and the allergen contribution to factor 1, and the y-axis represents the correlations between the variables and the allergen contribution to factor 2. Red lines represent the different allergens.

Figure 3

Immunoblotting results illustrated by plots of the serum samples showing the six reactive IgE-binding reactive bands. The reactive bands are represented by arrows and their estimated molecular weight. Panel A illustrates the immunoblot for the negative serum sample. Panel B shows a serum sample reactive to the P130 (gray arrow), P108 (green arrow), P83 (pink arrow), and P56 (purple arrow) IgE-binding protein bands; panel C illustrates a serum sample reactive to the P108 (green arrow), P83 (pink arrow), P56 (purple arrow), and P45 (black arrow); panel D demonstrates a serum sample reactive to the P145 (red arrow), P130 (gray arrow), P108 (green arrow), P83 (pink arrow), and P56 (purple arrow). MW (kDa) = molecular markers; on the left side of the panels we have blotted membranes, and on the right side, we have the blots from Image J Software. The y-axis in the plot represents the intensity of the reactive bands from the WB, illustrated as peaks, while the x-axis represents the distance in the membrane. The peaks in the plot match the bands in the membrane. Proteins (30μg crude MME) were separated in 4 – 15% gradient polyacrylamide gels. Sera dilution 1:400, Conjugate dilution 1:20000.

Figure 4

SDS-PAGE gel image analysis of the A. penicillioides crude mycelial mat extract. Different protein concentrations (lane 1: 1μg, lane 2: 3μg, and lane 3: 5μg) were separated on a 10% polyacrylamide gel and stained with SyproRuby (Invitrogen) for protein band pattern visualization and detection of the bands of interest (red arrows). MW (kDa) = molecular markers. Gel image created by the Proteomics and Mass Spectrometry Facility at Cornell University Institute of Biotechnology (Ithaca, NY).