Molecular and immunological characterization of allergens from the entomopathogenic fungus Beauveria bassiana

Abstract

Background: Entomopathogenic fungi such as Beauveria bassiana are considered promising biological control agents for a variety of arthropod pests. Beauveria species, however, have the potential to elicit allergenic reactions in humans, although no specific allergens have been characterized to date.

Methods: Four putative allergens were identified within B. bassiana expressed sequence tag (EST) datasets. IgE-reactivity studies were performed using sera from patients displaying mold allergies against recombinant B. bassiana proteins expressed in E. coli.

Results: Full length cDNA and genomic nucleotide sequences of four potential B. bassiana allergens were isolated. BLASTX search results led to their putative designation as follows; Bb-Eno1, with similarity to fungal enolases; Bb-f2, similar to the Aspergillus fumigatus major allergen, Asp f2 and to a fibrinogen binding mannoprotein; Bb-Ald, similar to aldehyde dehydrogenases; and Bb-Hex, similar to N-acetyl-hexosaminadases. All four genes were cloned into E. coli expression systems and recombinant proteins were produced. Immunoblots of E. coli extracts probed with pooled as well as individual human sera from patients displaying mould allergies demonstrated IgE reactivity versus recombinant Bb-Eno1 and Bb-Ald.

Conclusion: Four putative Beauveria bassiana allergens were identified. Recombinant proteins corresponding to two of the four, Bb-Eno1 and Bb-Ald were bound by sera IgEs derived from patients with fungal allergies. These data confirm the potential allergenicity of B. bassiana by identification of specific human IgE reactive epitopes.

Figure 1

SDS-PAGE analysis of B. bassiana recombinant proteins expressed in E. coli. SDS-PAGE, Coomasie Blue stained, extracts of E. coli strain BL21 harboring pRARE and the indicated expression plasmid constructs; lanes 1) and 2); pET41a:Bb-Eno1, lanes 3) and 4) pET41a:Bb-f2, lanes 5) and 6) pET41a:Bb-Ald, lanes 7) and 8) pET41a:Bb-Hex. Uninduced cell cultures, lanes 1), 3), 5), and 7). IPTG induced cell cultures, lanes 2), 4), 6), and 8).

Figure 2

SDS-PAGE analysis of soluble and pellet (inclusion bodies) fractions of the B. bassiana proteins expressed in E. coli. SDS-PAGE, Coomasie Blue stained extracts of soluble fractions lanes 1), 3), 5) and 7), and pellet fractions, lanes 2), 4), 6), and 8). Expression of Bb-Eno1, lanes 1) and 2), Bb-f2, lanes 3) and 4), Bb-Ald, lanes 5) and 6), and Bb-Hex, lanes 7) and 8).

Figure 3

SDS-PAGE analysis of the temperature sensitivity of the recombinant B. bassiana proteins. SDS-PAGE, Coomasie Blue stained, E. coli crude extracts subjected to; 1 min heat denaturation at 95°C, panel A), 5 min, 95°C, panel B), and 20 min, 95°C, panel C), Lanes correspond to crude extracts containing, lane 1) Bb-Eno1, lane 2) Bb-f2, lane 3) Bb-Ald, and lane 4) Bb-Hex.

Figure 4

Immunoblot analysis of recombinant B. bassiana proteins. Immunoblots were probed with sera pooled from (10 each) patients displaying mold allergies as indicated on the panels (A-J, and K-T). The final concentration of individual sera in each pool was 1:35. An HRP conjugated goat anti-human IgE antibody was used as the secondary antibody. Lanes contain recombinant E. coli expressed proteins as follows, lane 1) Bb-Eno1, 2) Bb-f2,. 3) Bb-Ald, 4) Bb-Hex. Lane 5) 40 μg crude B. bassiana extract.

Figure 5

Immunoblot analysis of recombinant B. bassiana proteins. Immunoblots were probed with sera pools (2 each) as indicated on the panels (AB, CD, EF, GH, and IJ), with a 1:5 final concentration of individual sera in each pool. Blots were probed with an HRP conjugated goat anti-human IgE antibody as the secondary antibody. Lanes contain recombinant E. coli expressed proteins as follows, lane 1) Bb-Eno1, 2) Bb-f2,. 3) Bb-Ald.

Figure 6

Immunoblot analysis of recombinant Bb-Ald. PVDF membrane strips containing crude extracts of E. coli expressed Bb-Ald were probed with 1 mL of each designated sera pool, with each pool containing two sera (final dilution 1:5 each sera). Arrow indicates the position of Bb-Ald.

Figure 7

Ponceau S staining and immunoblot analysis of recombinant B. bassiana. Immunoblots were probed with individual sera, A) and B) as indicated on the panels using a 1:5 final concentration of sera. Blots were probed with an HRP conjugated goat anti-human IgE antibody as the secondary antibody. Lanes contain recombinant E. coli expressed proteins as follows, lane 1) Bb-Eno1, 2) Bb-f2,. 3) Bb-Ald, and 4) Bb-Hex. Panel 1) represents Ponceau S staining of the PVDF membrane after transfer.

Figure 8

Full length amino acid sequences of 24 enolases deposited in the NCBI Genbank database were used to construct an enolase phylogram. Normalized posterior probabilities values greater than or equal to 0.9 are presented at their respective nodes. Known allergenic enolases are denoted by an asterisk.