Polymorphism of Aspergillus Fumigatus Major Allergen Genes Associating with Their Isolated Sites Affects Their IgE Epitope Structures


 The circumstances in which organisms live induce polymorphism in their genes, including fungal allergen genes, leading to altered structures and functions of proteins, related to their pathogenicity. Major allergen genes of Aspergillus fumigatus, Asp f 1, Asp f 2, and Asp f 3, were examined in 59 strains [environment and animal/human-body origin] to determine their nucleotide sequences, and then categorized. The location and number of IgE epitopes on the allergen molecules were predicted using a computer software. The Asp f 1 gene was classified into two groups (f1-1 and f1-2). One of the groups possessed one-nucleotide mutation point with one amino-acid substitution. The mutated Asp f 2 gene accompanying 6-amino acid substitution was classified into 7 groups (f2-1 to f2-7). Six of the groups possessed a newborn IgE epitope. The Asp f 3 gene contained two mutations, resulted in three groups (f3-1 to f3-3) without any amino-acid substitutions. Category E, consisting of groups f1-1, f2-5, and f3-2, was specific to an environmental origin. Our findings suggest that nucleotide mutation of the fungal allergen genes, associated with the origin of the fungus, modifies the structure of proteins, and affects their pathogenic properties, such as the localization of IgE epitopes.


Introduction
Fungi cause human diseases, such as infections, mycotoxicosis, and allergies. It is believed that fungi are ubiquitous airborne allergens. More than 80 fungi genera are recognized to be associated with allergenic symptoms 1 . The genera Alternaria, Cladosporium, Penicillium, Aspergillus, and Malassezia has been well studied 2 . A. fumigatus is ranked the top pathogenic fungus. A.fumigatus causes infections and allergies, such as allergic rhinitis, asthma, sinusitis, and allergic bronchopulmonary aspergillosis (ABPA) 3,4 .
Allergenic proteins are de ned as proteins that are capable of stimulating the production of IgE antibodies in the human body and to speci cally bind to IgE antibodies, which exist in the sera of allergic patients. A candidate protein is identi ed as an allergen after epidemiological, etiological, and immunological examinations. Once the gene of such protein is cloned, and sequenced, it is registered in the International Union of Immunological Societies allergen database (IUIS allergen database; URL http://www.allergen.org/).
A. fumigatus allergenicity is also well analyzed 1,2 , and a total of 23 A. fumigatus allergens are listed in the IUIS allergen database. Several of these, namely Asp f 1, Asp f 2, and Asp f 3, are recognized as major A. fumigatus allergens [5][6][7][8] . A total of 85% of patients with ABPA or asthma who showed a positive immunoglobulin E (IgE) antibody titer against the extracted allergens of A. fumigatus possessed an IgE antibody against the puri ed and recombinant Asp f 1 protein 6 . Asp f 2 is an allergic protein with a molecular weight of 37 kDa. Both isolated and recombinant Asp f 2 protein bound to the IgE antibody in sere of ABPA and cystic brosis-ABPA patients 7 . Asp f 3 allergen, which was cloned in 1997, is a protein composed of 168 amino acids. The binding ability of IgE antibodies to recombinant Asp f 3 was detected in 72% of 89 patients who were positive for a skin test using the crude allergen extracted from an A.fumigatus culture 8 .
Infection causes modi cations in the cellular and molecular events occurring between hosts and pathogens. Such interactions may be one of the pressures that leads to many kinds of alterations of organism properties, via nucleotide mutation of genes. In addition, the origin of microorganisms is an important factor in mutations. Environmental pressure induces epigenetic and genetic modi cations on all creatures, including fungi.
We are interested in the in uence of the origin of the isolated fungi on the genes of allergens, which affect the immunological conditions of humans. The objective of this study was to examine the nucleotide mutations in allergen genes, Asp f 1, Asp f 2, and Asp f 3, of A. fumigatus isolated from the environment, such as house dust, room air, and soil, and from the body of animals/humans, resulting from the colonization of A. fumigatus after infection. We discuss the probability that the origin-speci c adaption of allergen genes makes fungus more allergenic.

Results
Polymorphic properties of Aspergillus fumigatus allergen genes, Asp f 1, Asp f 2, and Asp f 3.
A total of 57 strains (96.6%) of the 59 isolates showed the sequence of Asp f 1 gene, same as that registered in the IUIS database. The other two strains of the Asp f l gene showed one nucleotide mutation at the 262 bp position (Table 1). This mutation resulted in an amino acid substitution at the 88th residue. These data indicate that the Asp f 1 gene is classi ed into two groups: f1-1 and f1-2 ( Table 1).
The sequencing of the tested strains of the Asp f 2 gene indicated 10 mutation points accompanying amino acid substitutions ( Table 1). The nucleotide sequence of Asp f 2 gene in the IUIS database was not found among any of the 59 strains tested. The Asp f 2 gene of the 59 strains was classi ed into seven groups, based on the combination of mutation points (Table 1). A total of 42 of the 59 strains (71.2%) belonged to the f2-2 group. This dominant group contained four mutation points, which were associated with three amino acid substitutions ( Table 1). The f2-5 group (12 strains, 20.0%) was the next dominant group. It contained one more mutation point than the f2-2 group.
The Asp f 3 gene of the 59 strains showed three distinct nucleotide sequences. A total of 18 (30.5%) of the 59 strains corresponded to the nucleotide sequence of the Asp f 3 gene registered in the IUIS database (group f3-1). Of the 59 strains, 34 (57.6%) showed one mutation in the sequence at 147 bp (group f3-2). This mutation group was dominant. Group f3-3 possessed one more mutation point than group f3-2, which was a mutation at 240 bp ( Table 1). Neither of these two nucleotide mutations affected the amino acid sequence.
Categorization of the groups of the allergen genes of Aspergillus fumigatus and their relationship to their origin.
Categories A to G were recognized based on a combination of the groups of Asp f 1, Asp f 2, and Asp f 3 in the total of 42 strains whose origin was clari ed ( Table 2). Categories C, D, F, and G were minor because of the markedly small number of strains, suggesting the importance of Categories A, B, and E.
Categories A and B consisted of the isolates whose origin was environment or disease (animal/human body). However, category E consisted of isolates derived from the environment ( Table 2).
All isolates of A. fumigatus were classi ed, according to the category and group in Table 3. Group f3-2 existed in categories B and E. Group f3-2 was related to group f2-2 in category B, and with f2-5 in category E. Group f2-2 in category B consisted of the isolates from disease and environment origins, but group f2-5 speci cally consisted of the isolates from the environment origin.
The in uence of amino-acid substitution of Aspergillus fumigatus allergen proteins on their IgE epitopes.
The properties of Asp f 1 protein, regarding the amino-acid substitution resulting from polymorphisms of the nucleotide sequence and the information for its IgE epitopes are summarized in Fig. 1. The dominant f1-1 group (57 of 59 strains), which possessed a sequence that was the same as that registered in the IUIS database, contained ve IgE epitope sites (Fig. 1, panel B) on the Asp f 1 molecule. The two stains (3.4%) that belonged to the f1-2 group included a mutated nucleotide sequence, resulting in an amino acid substitution at the 88th position and a deletion of the IgE epitope ( Fig. 1, panel B). The threedimensional structure is shown in Fig. 4, panel D.
The properties of the Asp f 2 allergen, regarding the amino acid substitution and the information for its IgE epitope sites are summarized in Fig. 5. Although the nucleotide mutation occurred at 10 positions of the Asp f 2 gene, four of these mutations did not in uence the amino acid sequence. The other 6 mutations, however, replaced the amino acids. One strain of the f2-1 group, in which there was no replacement at position 297th, revealed four IgE epitopes (Fig. 2, epitopes A to D). Group f2-2, which was dominant (42 of 59 stains), contained ve IgE epitope sites (Fig. 5, epitopes A to E). Although the amino acid sequences of the f2-3, f2-4, f2-5, f2-6, and f2-7 groups were different from that of the dominant f2-2 group, the construction properties of the IgE epitopes were the same as those of group f2-2. A new epitope (E) was induced in the Asp f 2 protein of group f2-5 (Fig. 2, panels B to D).
The properties of the Asp f 3 allergen protein, regarding the construction of its IgE epitopes, are summarized in Fig. 3. Although polymorphism in the nucleotide sequence of the Asp f 3 gene was detected, no amino-acid substitutions occurred. All of the strains contained two IgE epitope sites (Fig. 3, panels B to D).

Discussion
Nucleotide mutations of A. fumigatus allergen genes, Asp f 1, Asp f 2, and Asp f 3, were studied. Asp f 1 gene was classi ed into two groups, f1-1 and f1-2 according to the mutations ( Table 1). The sequence of group f1-1 was the dominant one, and was completely identical to that registered in the IUIS allergen database. A single nucleotide polymorphism of the Asp f 2 gene led to 7 different genetic groups (f2-1 to f2-7) ( Table 1). Among the sequences of the Asp f 2 gene, the sequence registered in the IUIS allergen database was not found in the tested strains. The Asp f 3 gene was classi ed into three groups (f3-1 to f3-3) based on its nucleotide polymorphisms ( Table 1). The sequence of the Asp f 3 of the f3-1 group was in complete accordance with that registered in the IUIS database. One nucleotide mutation in the f3-2 group and two mutations in the f3-3 group occurred. Categories A to G were provided, depending on the grouping of the three allergen genes of A. fumigatus (Table 2). In particular, categories A, B, and E were the major concerns. Categories A and B existed in both the environment and disease (animal/humanbody) origins, suggesting no origin-speci city. However, category E was markedly speci c to the environment ( Table 2). Category E consisted of groups f1-1, f2-5, and f3-2. Group f1-1 was distributed into all A. fumigatus strains, and group f3-2 was recognized in strains derived from the environment and animal/human-body. However, group f2-5 was the particular one found in the environment. This fact indicates that a particular mutation of the nucleotide, depending on its origin, relates to a stable settlement to its living circumstances. It is believed that each A. fumigatus strain has adapted to its individual situation, accompanied by the mutation of some genes, such as allergen genes.
Two functional IgE epitopes were experimentally identi ed on the Asp f 1 molecule [12]. One of the epitopes was located from the 7th to the 22nd amino-acids from the N-terminal end. The other was located from the 140th to the 149th amino-acids [13,14]. The Asp f 1 gene mutation described in this study resulted in an amino-acid substitution (Table 1, Fig. 1). When an analysis was performed using computer software to predict IgE epitopes, ve epitopes existed in the Asp f 1 allergen of group f1-1 (Fig.  1). The mutation in the nucleotides of group f1-2 resulted in the deletion of one epitope (Fig. 1, epitope E), but such allergological modulation is almost negligible due to the small number of isolates present in group f1-2. This implies that the nucleotide sequence of Asp f 1 registered in the IUIS allergen database is unquestionably available for use in allergen research, such as the preparation of recombinant Asp f 1 proteins.
The nucleotide sequence of Asp f 2 registered in the IUIS allergen database was not found in any of the strains tested, suggesting that the registered sequence would not be suitable for allergen research. A single nucleotide polymorphism of the Asp f 2 gene induced two allergological types. One is based on the sequence of group f2-1, but the signi cance of the presence of group f2-1 would be negligible because of the markedly small number of isolates present in group f2-1. The other type consists of groups f2-2 to f2-7 (Fig. 5, B). The epitope site at 155 YTTRR 159 was experimentally determined [14]. This epitope might be affected by the amino-acid substitution at the 159th position. On the other hand, the amino-acid substitution at the 297th position generated a new IgE epitope (Fig. 2, epitope E, panels B, C, and E). As mentioned above, group f2-5, which possesses a newborn IgE epitope, is markedly speci c to the origin of the environment. It would be possible to consider that A. fumigatus strains belonging to group f2-5 were mutated and adapted to the environment, accompanied by the acquisition of a new IgE epitope. A. fumigatus strains in group f2-5 might be more allergic than others.
The Asp f 3 gene was classi ed into 3 groups based on the nucleotide polymorphism. The sequence of Asp f 3 of the f3-1 group was in complete accordance with that registered in the IUIS database. One nucleotide mutation in the f3-2 group and two mutations in the f3-3 group were found. These two nucleotide substitutions did not induce any amino-acid substitutions. A conformational IgE epitope was experimentally determined in the Asp f 3 molecule [15]. The amino-acid sequence of Asp f 3 is appropriate for any kind of allergen research because there are no amino-acid substitutions.
It is known that the in uenza virus mutates depending on its origin, accompanied by an increased pathogenicity [16,17]. The Asp f 2 allergen of group f2-5 possesses one more epitope, compared with the other genetic type, suggesting its facilitated pathogenicity. These ndings presume that the isolates of A. fumigatus belonging to Category E would adapt to circumstances, such as the environment, via the mutations in the Asp f 2 gene. Category E was markedly speci c to the environmental origin. As mentioned above, group f2-5 was strictly related to group f3-2, which was the dominant group in Asp f 3.
Although there is still no explanation for how the Asp f 2 gene of the f2-5 group relates to the Asp f 3 gene of group f3-2 described in this communication, the relationship between the adaptation of fungi to the environment and the distribution of IgE epitopes on allergic proteins is still interesting.

Conclusions
The polymorphism of A. fumigatus allergen genes occurred via mutations, and this polymorphism was related to the circumstances from which strains were isolated. The mutated nucleotide sequences affected their protein properties, accompanied by an IgE epitope structure. In pathogenic viruses, an increase in pathogenicity occurs due to mutations related to their origins. Our ndings suggest that nucleotide mutation of fungal allergen genes, associated with the origin from which the fungus was isolated, modi es the structure of the proteins, affecting their pathogenic properties, such as their IgE epitope localization.

Fungal strains.
A total of 59 strains of A. fumigatus, including 29 strains from different environments [from room air (2), house dust (12), and soil (15)], 13 from infectious lesions [from humans (6) and animals (7)] and 17 of unknown source, were used in this study. The strains were collected in several area in Japan, and sent to, and maintained in the mycological laboratory of the National Institute of Health Sciences (Kawasaki, Japan).
Identi cation of the fungal isolates.
All stocked isolates were inoculated on malt extract agar (Oxoid, Thermo Scienti c, Wilmington, DE) and Czapek agar containing 3.5% Czapek-dox broth (Becton, Dickinson and Company, Franklin Lakes, NJ) and 1.5% agar, and incubated at 25°C for 10 days. The Aspergillus strains were examined and identi ed using the general method by Raper et al. [9], with a focus on morphology.
All strains were cultured on a potato dextrose agar (Eiken, Tokyo, Japan) slant medium at 25°C for 10 days. Mycelia or conidia were harvested from the slant culture in a tube containing 1 ml of potato dextrose broth (Becton, Dickinson and Company), and incubated at 25°C for 3 days. These cultures were centrifuged at 18,000×g for 10 min. Genomic DNA was extracted from the pellets using the sodium dodecyl sulfate (SDS) method with minor modi cations [10]. Brie y, an 1-ml aliquot of a lysing buffer containing 50 mM Tris-HCl buffer (pH 8.0), 250 mM NaCl, 50 mM EDTA, 0.3% SDS, and 20 μl of RNase A (10 mg/ml; Novagen, Darmstadt, Germany) were added to the tube containing the fungal body pellets, and mixed vigorously. The upper water-phase containing genomic DNA was harvested. The genomic DNA was precipitated using chilled isopropanol, and suspended in 20 μl of Tris-EDTA buffer, (pH 7.5). The extracted genomic DNA was measured spectrophotometrically to examine its quality and concentration using a NanoDrop 1000 Spectrophotometer V3.7 (Thermo Fisher Scienti c, Wilmington, DE).
The β-tubulin gene was used in this study as the identi cation marker gene of A.fumigatus. A fragment of the β-tubulin gene was ampli ed using the primer pair Bt2a (5'-GGTAACCAAATCGGTGCTGCTTTC-3') and Bt2b (5'-ACCCTCAGTGTAGTGACCCTTGGC-3') [11]. TaKaRa ExTaq (TaKaRa Bio Inc., Otsu, Japan) was used according to the manufacturer's instructions for ampli cation in a thermal cycler (GeneAmp PCR System 9700; Applied Biosystems, Foster City, CA). The PCR program consisted of an initial denaturing step at 94 °C for 5 min, 35 ampli cation cycles (94°C for 30 sec, 60°C for 40 sec, and 72°C for 1 min), and an additional extension step at 72°C for 3 min. The PCR products were puri ed using ExoSap-IT (USB; Cleveland, OH), and then directly sequenced. The obtained two sequences were concatenated into one sequence using a sequence assembly software program, ATGC (Genetyx Corporation, Tokyo, Japan). The determined sequences were entered into the Basic Local Alignment Search Tool (BLAST) for comparison with the sequences registered in the nucleotide database of the National Center for Biotechnology Information. When the result of a sequence homology search of the isolates indicated a high homology with the A.fumigatus β-tubulin gene, the isolate was identi ed as A. fumigatus.
The determination of the nucleotide sequence of Aspergillus fumigatus allergen gene.
The fragments of these genes that contained a coding region sequence were ampli ed and sequenced using the following primer pairs, which were designed using a publicly available software program, Primer 3 Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/), based on the sequences of Accession Nos. M83781 (Asp f 1), U56938 (Asp f 2), and U58050 (Asp f 3).