Thioredoxin from the Indianmeal moth Plodia interpunctella: cloning and test of the allergenic potential in mice

Abstract

Background/objective: The Indianmeal moth Plodia interpunctella is a highly prevalent food pest in human dwellings, and has been shown to contain a number of allergens. So far, only one of these, the arginine kinase (Plo i 1) has been identified.

Objective: The aim of this study was to identify further allergens and characterise these in comparison to Plo i 1.

Method: A cDNA library from whole adult P. interpunctella was screened with the serum of a patient with indoor allergy and IgE to moths, and thioredoxin was identified as an IgE-binding protein. Recombinant thioredoxin was generated in E. coli, and tested together with Plo i 1 and whole moth extracts in IgE immunoblots against a large panel of indoor allergic patients' sera. BALB/c mice were immunised with recombinant thioredoxin and Plo i 1, and antibody production, mediator release from RBL cells, T-cell proliferation and cytokine production were measured.

Result: For the first time a thioredoxin from an animal species was identified as allergen. About 8% of the sera from patients with IgE against moth extracts reacted with recombinant P. interpunctella thioredoxin, compared to 25% reacting with recombinant Plo i 1. In immunised BALB/c mice, the recombinant allergens both induced classical Th2-biased immune responses such as induction IgE and IgG1 antibodies, upregulation of IL-5 and IL-4 and basophil degranulation.

Conclusion: Thioredoxin from moths like Plo i 1 acts like a classical Type I allergen as do the thioredoxins from wheat or corn. This clearly supports the pan-allergen nature of thioredoxin. The designation Plo i 2 is suggested for the new P. interpunctella allergen.

Figure 1. IgE immunoblots with patients' sera.

Top panels: extracts from whole mediterranean flour moths (E. kuehniella) were separated by polyacrylamide electrophoresis, blotted onto nitrocellulose, and probed with the sera from various groups of patients with indoor allergy or seafood allergy as well as from control individuals. The indoor allergic patient groups were: M, housedust mite allergic patients (n = 69), M+D, patients allergic to mites and animal dander (n = 43), D−M, patients with allergy to animal dander but not to mites (n = 20). Patients SF, seafood allergic patients (n = 22), NC, nonallergic control individuals (n = 2), BC, buffer control without serum, only secondary anti-IgE antibody. Molecular weights [kDa] are indicated at the left side. Bottom panels, the recombinant P. interpunctella 40 kDa arginine kinase (AK) and 12 kDa thioredoxin (TRX) were probed with those patients' sera containing IgE to E. kuehniella, and only the parts of the strips containing the two recombinant allergens are shown.

Figure 2. Comparison of thioredoxin sequences.

The deduced amino acid sequence of P. interpunctella thioredoxin Plo i 2 (Pi) was aligned with Bombyx mori thioredoxin (ABM92269) (Bm) with 83% identity. The other arthropod thioredoxin from the shrimp Litopenaeus vannamei (ACA60746) (Lv) was 58% identical, and human thioredoxin (NP_003320) (Hs) shared 47% amino acid identity with Pi. The allergenic fungal thioredoxin (2J23_A) Mala s 13 from Malassezia sympodialis (Ms) and wheat (Triticum aestivum) thioredoxin Tri a 25 (Ta) (CAB96931) were 46% and 41% identical, respectively with Plo i 2. The residues equal to Plo i 2 are marked with dashes. The important thioredoxin consensus sequence WCGPC with the two active site cysteines was found in all the sequences (bold type).

Figure 3. Immunoblot inhibition experiment.

Extracts from E. kuehniella were separated by polyacrylamide electrophoresis, blotted onto nitrocellulose, and strips were probed with serum samples preincubated with or without 10 µg of recombinant allergen overnight. Lane 1, thioredoxin (Plo i 2) positive serum; lane 2, same serum preincubated with recombinant P. interpunctella thioredoxin; lane 3, arginine kinase (Plo i 1) positive serum; lane 4, same serum preincubated with recombinant Plo i 1; lane 5, nonallergic control serum, lane 6, same serum preincubated with Plo i 2; lane 7, same serum preincubated with Plo i 1; lane 8, buffer control. Molecular weights [kDa] are indicated at the left side, the positions of thioredoxin (TRX) and arginine kinase (AK) at the right side.

Figure 4. Antibody levels in mouse sera.

(A), thioredoxin, (B), arginine kinase-specific antibody responses in sera after immunisation with three different antigen concentrations (1 µg, 5 µg, 25 µg) measured by ELISA; (C), IgE-mediated basophil degranulation in sera, β- hexosaminidase release from thioredoxin- and arginine kinase-sensitised mice, treated with 1 µg (black bars), 5 µg (grey bars) or 25 µg (striped bars), compared to sham-treated controls (white bars). *p<0.05, **p<0.01, ***p<0.005.

Figure 5. Cellular immune responses.

IL-5, IL-4 and IFN-γ levels in supernatants of restimulated spleen cell cultures of mice, treated with 1 µg (black bars), 5 µg (grey bars) or 25 µg (striped bars) of (A), thioredoxin or (B), arginine kinase, or sham-treated (white bars). Groups were compared to sham-treated controls. *p<0.05, **p<0.01.

Figure 6. In vitro splenocyte stimulation.

Stimulation index [SI] of thioredoxin or arginine kinase restimulated spleen cell cultures of mice, treated with 1 µg (black bars), 5 µg (grey bars) or 25 µg (striped bars) antigen, compared to sham-treated controls (white bars). **p<0.01.