The emerging pathogen Paecilomyces variotii ‐ a novel and important fungal allergen source

To the Editor, Fungal allergy represents a worldwide public health burden.1 Owing to their ability to colonize and germinate in the respiratory mucosa, fungi can have a far greater impact on the patients' immune system than other respiratory allergen sources.1 However, the lack of knowledge about allergenic fungi is one of the factors that contribute to a general underdiagnosis of fungal allergy.2 In the present study, we investigated the allergenic potential of Paecilomyces variotii, a heatresistant mold ubiquitously found in soil, decomposing organic material, food products, and clinical samples, that is regarded as an emerging cause of serious, sometimes lifethreatening, infections, and is often detected in the indoor environment.3– 6 Even though P. variotii has already been suggested as an aeroallergen source and occupational hazard, no allergens have yet been identified or characterized by this species.7 IgE immunoblots, performed with four different pools of sera from moldsensitized patients (Table S1), showed the presence of several IgEreactive proteins in P. variotii extract (Figure 1A), suggesting the species’ high allergenic potential (no reaction was observed in immunoblots incubated with the detection antibody only— data not shown). Mass spectrometry analysis led to the identification of the first three P. variotii allergens, which interestingly all represent enzymes. Protein A (~36 kDa) was identified as a transaldolase, an enzyme that is already known as an allergen in different fungal species.8 Protein B (~37 kDa) was identified as a glyceraldehyde 3phosphate dehydrogenase, which represents an important allergen in various sources,9 and Protein C (~48 kDa) was identified as an enolase. Enolases have also already been identified as highly conserved and crossreactive allergens in various allergen sources including different fungi.10,11 The proteins were submitted to the WHO/IUIS Allergen Nomenclature Subcommittee and were tentatively assigned the allergen names Pae v 1a, Pae v 2b, and Pae v 6c. The IgEreactive protein bands marked D to H still have to be identified by peptide mass fingerprinting. Fulllength cDNAs coding for the P. variotii proteins Pae v 1, Pae v 2, and Pae v 6 were generated, cloned into a bacterial expression vector, and recombinant proteins were produced in Escherichia coli with a Cterminal hexahistidinetag. Reducing and nonreducing SDSPAGEs (Figure S1) and immunoblots, performed with an antihistidinetag antibody, verified the purity of the recombinant proteins and showed that all proteins possess the capability to form disulfide bridges and to oligomerize under nonreducing conditions (Figure S1). Moreover, circular dichroism spectroscopy analysis of the recombinant allergens showed farUV spectra with broad minima between 210 and 220 nm, which suggested the presence of both, alphahelical structures, and betasheets, and indicated that the proteins represent properly folded molecules (Figure S2). ELISAs performed with sera from moldallergic patients showed the IgEbinding capacity of the three recombinant proteins. With OD values of more than 3.0 (Figure 1B), the enolase rPae v 6 showed the highest levels of IgE reactivity, whereas the IgE reactivities of rPae v 1 (maximum OD value of 0.8) and rPae v 2 (maximum OD value of 0.390) were considerably lower. Furthermore, IgE inhibition immunoblots, where nitrocelluloseblotted protein extracts from P. variotii were exposed to serum pools that had been preincubated with either one of the recombinant proteins or, for control purposes, with buffer only, showed that the recombinant allergens rPae v 1, rPae v 2, and rPae v 6 were able to significantly reduce patients' IgEbinding to their natural counterparts (Figure 1C). This indicates that the recombinant molecules contain the IgEbinding epitopes of their natural counterparts and suggests that rPae v 1, rPae v 2, and rPae v 6 could be used as tools for in vitro diagnosis of P. variotii sensitization. Preincubation with rPae v 2, a GAPDH, caused the reduction of IgEbinding to several proteins with molecular weights of 25– 45 kDa. This suggests the presence of different Pae v 2 variants with different posttranslational modification and Pae v 2 degradation products in the natural fungal extract. The presence of GAPDH variants, which can also influence the molecule's different functions, has already been suggested in the literature.12 Analysis of the prevalence of IgE recognition in a cohort of patients, sensitized to different mold species, by ELISA revealed different frequencies of IgE reactivity. In Figure S3, the presence (in green) or the absence (in red) of IgEbinding to the recombinant allergens is displayed for each patient. rPae v 1 was recognized by 22% (11 out of 49) of the tested patients, rPae v 2 by 6% (3 out of 50), and 68% of the tested patients (39 out of 57) reacted with rPae v 6. Among the patients tested with all three recombinant allergens, rPae v 1 was recognized by 22% (9 out of 41), rPae v 2 by 5% (3 out of 41), and rPae v 6 by 61% (25 out of 41) of the patients. Based on these results, rPae v 1 and rPae v 2 can be regarded as minor allergens of P. variotii, whereas