Novel Cytosolic Allergens of Aspergillus fumigatus Identified from Germinating Conidia

Abstract

Aspergillus fumigatus is the common cause of allergic broncho-pulmonary aspergillosis (ABPA) and most of the allergens have been described from its secreted fraction. In the present investigation, germinating conidial cytosolic proteins of A. fumigatus were extracted from a 16 h culture. The proteome from this fraction was developed, and immuno-blots were generated using pooled ABPA patients' sera. Well separated Immunoglobulin-E (IgE) and Immunoglobulin-G (IgG) reactive spots were picked from corresponding 2DE gels and subjected to mass spectrometric analysis. As a result, 66 immuno-reactive proteins were identified from two geographically different strains (190/96 and DAYA) of A. fumigatus. Only 3 out of 66 proteins reacted with IgG, and the remaining 63 proteins were found to be IgE reactive. These 63 IgE-reactive cytosolic proteins from germinating conidia included 2 already known (Asp f12 and Asp f22) and 4 predicted allergens (Hsp88, Hsp70, malate dehydrogenase, and alcohol dehydrogenase) based on their homology with other known fungal allergens. In view of this, the panel of presently identified IgE-reactive novel proteins holds the potential of providing a basis for the wider diagnostic application in assay for allergic aspergillosis. We could demonstrate that recombinantly expressed proteins from this panel showed consistent reactivity with IgE of individual sera of ABPA patients. The recombinantly expressed proteins may also be useful in desensitization therapy of allergic disorders including ABPA.

Spectrum of Allergens and Allergen Biology in India

“…Different species of Aspergillus are highly predominant in the ambient air of India and are major contributing factors for the development of asthma as they discharge a huge number of allergen-containing spores [83]. Two separate immunoproteomic investigations [84, 85] were carried out in which several new, as well as previously unreported immunoreactive molecules, were identified from the intercellular as well as extracellular (secretome) proteome of A. fumigatus . Most of these antigenic molecules displayed strong IgE and IgG responses in asthmatics and aller gic broncho-pulmonary aspergillosis (ABPA) patients.…”

Proteomic analysis of Aspergillus fumigatus – clinical implications

“…Shotgun proteomics has facilitated analysis of proteins previously intractable to 2-DE, such as membrane proteins through gel-free approaches [15,16]. However, 2-DE still plays an important role in analysing post-translational modifications and the immunoproteome [17][18][19][20][21][22][23][24].…”

“…Recent work has used quantitative shotgun proteomics to provide a detailed insight into stage-specific proteomic events relevant to early host colonisation [30,31]. Cagas [19,25,29,30]. 143 proteins were detected in all time points, many of which are conserved in other fungi.…”

New Commercially Available IgG Kits and Time-Resolved Fluorometric IgE Assay for Diagnosis of Allergic Bronchopulmonary Aspergillosis in Patients with Cystic Fibrosis

“…Apart from Man1P, these proteins were also identified by Singh et al using an immunoproteomic approach, including sorting by IgE Western blot analysis with sera from ABPA patients (6,18). Two of them, G6Pi and NAD, were highlighted as ABPA specific (6,18).…”

“…For this study, we developed an in-house DELFIA to measure IgE levels against several Aspergillus antigens (A. fumigatus purified protein extract [PPE] and five recombinant antigens, including those previously highlighted by Singh et al [6,18]). …”