Group Title: Clinical and Molecular Allergy 2006, 4:12
Title: Molecular and immunological characterization of allergens from the entomopathogenic fungus Beauveria bassiana
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Title: Molecular and immunological characterization of allergens from the entomopathogenic fungus Beauveria bassiana
Series Title: Clinical and Molecular Allergy 2006, 4:12
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Creator: Westwood GS
Huang SW
Keyhani NO
Publication Date: 38982
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Clinical and Molecular Allergy Bi



Research

Molecular and immunological characterization of allergens from
the entomopathogenic fungus Beauveria bassiana
Greg S Westwood', Shih-Wen Huang2 and Nemat O Keyhani*1

Address: 'Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA and 2Department of Pediatrics,
University of Florida, College of Medicine, 32610, USA
Email: Greg S Westwood gregwest@ufl.edu; Shih-Wen Huang huangsw@peds.ufl.edu; Nemat O Keyhani* keyhani@ufl.edu
* Corresponding author


ed Central

O^^


Published: 22 September 2006
Clinical and Molecular Allergy 2006, 4:12 doi: 10.1 186/1476-7961-4-12


Received: 01 August 2006
Accepted: 22 September 2006


This article is available from: http://www.clinicalmolecularallergy.com/content/4/1/12
2006 Westwood et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



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-Enol, 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-Enol and Bb-Ald.
Conclusion: Four putative Beauveria bassiana allergens were identified. Recombinant proteins
corresponding to two of the four, Bb-Enol 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.


Background
Allergic diseases represent a growing human health prob-
lem, affecting up to 25% of individuals living in industri-
alized nations [1]. Both in- and outdoor populations of
filamentous fungi are a major cause of human allergies
and asthma, and can in some cases, lead to severe allergic
disease [2]. Overall, some 30% of asthma cases can be


attributed to exposure and sensitization to filamentous
fungal allergens [3-5].

Beauveria bassiana is an entomopathogenic fungi currently
under intensive study as a biological control agent against
a wide range of agricultural, nuisance, and disease carry-
ing insect pests [6-10]. B. bassiana is considered non-path-


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Clinical and Molecular Allergy 2006, 4:12


ogenic to vertebrates, has not been deemed a potential
health or environmental hazard [11], and has received
EPA approval for commercial use. Volumetric assays of
allergens performed in the Netherlands in the 1980's,
revealed that although the environmental concentration
of Beauveria spores was very low, the allergic response was
quite high [12,13]. Using skin prick assays on patients
with mold allergies, B. bassiana was shown to elicit one of
the strongest reactions relative to the other fungal species
tested. More recently, it has been confirmed that crude
extracts of B. bassiana can elicit allergic reactions in
humans [14]. Sera IgEs derived from patients displaying
allergies to molds as well as from people with no known
allergies reacted with several proteins present in B. bassi-
ana crude extracts. Many of these proteins were cross reac-
tive with epitopes present in a number of major allergenic
fungi, however the identities of any specific B. bassiana
allergen has yet to be reported. In order to gain more
information concerning B. bassiana and its potential aller-
genicity it is important to isolate the genes coding for IgE-
binding allergens and characterize their protein products.
Recombinant purified allergens, as compared to crude
fungal extracts, can then be used to examine the nature of
the IgE binding as well as in the diagnosis of allergy, in
that the recombinant proteins are more standardized, can
be highly purified, and hence are more suitable for immu-
nodiagnosis [15,16].

A significant number of fungal allergens are proteins of
unknown function, although the biochemical activities of
a number of allergens have been characterized. These typ-
ically fall into several classes including metabolic
enzymes, proteases, and enzyme inhibitors [5,17]. A mol-
ecule identified as an allergen in one species of fungus is
often found to be an allergen when identified in other
species, presumably due to similarities in structure and
hence IgE-reactive epitopes. Thus, aldehyde dehydroge-
nase has been identified as an allergen in both Alternaria
alternate (Alt al0) and Cladosporium herbarum (Cla h3)
[18]. Amongst other metabolic enzymes, enolases (2-
phosho-D-glycerate hydrolase) from a wide range of
organisms, are common allergens with shared epitopes
[19-21]. This phenomenon of cross-reactivity of an IgE
produced in response to an antigen from one organism to
another can lead to wide spectrum allergic reactions
derived from the original sensitization [22-24].

Here we report the identification of four B. bassiana pro-
teins as potential allergens. Full length cDNA and
genomic nucleotide sequences of the four genes were
determined. Similarity search results of the translated
open reading frames of the proteins coded by the genes
have led to their putative designation as follows; Bb-Eno 1,
an enolase; Bb-Ald, aldehyde dehydrogenase; Bb-f2, simi-
lar to Asp f2 and a fibrinogen binding mannoprotein; and


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Bb-Hex, an N-acetylhexosaminidase. The cDNA
sequences of the proteins were used to design primers for
subcloning of the genes into E. coli expression vectors. All
four proteins were expressed as recombinant proteins in
E. coli. Two of these proteins, Bb-Enol and Bb-Ald reacted
with human IgEs derived from patients displaying mold
allergies.

Methods
Strains and cultures
Beauveria bassiana (ATCC 90517) was maintained on
Potato dextrose (PD) agar at 26 C. E. coli stains TOPO
ToplO (Invitrogen, CA) and BL21 Rosetta (DE3), harbor-
ing the pRARE plasmid (Novagen, Darmstadt, Germany)
were used for routine cloning and protein expression,
respectively. E. coli strains were grown in Luria-Bertani
(LB) nutrient broth or agar plates supplemented with the
appropriate antibiotics as indicated.

Bioinformatic identification of putative allergen genes
Construction and sequencing of expressed sequence
tagged (EST) cDNA libraries derived from five different
developmental stages of B. bassiana has recently been
reported [25,26]. Additional sequences were obtained by
suppressive subtractive hybridization (SSH) using fungal
cells grown on insect cuticles and fungal cells grown on
glucose as the tester and driver mRNAs respectively using
established protocols [27,28]. BLASTX similarity searches
using the sequence dataset (~18,000 ESTs) revealed four
sequences with high homology to allergen genes.

Molecular manipulations
Molecular manipulations including plasmid isolation,
restriction digestion, agarose-gel electrophoresis, and PCR
were performed using standard methods. Template
mRNA was extracted from B. bassiana grown on minimal
medium (per L; 0.4 g KH2PO4, 1.4 g Na2HPO4, 0.6 g
MgSO4-7H20, 1.0 g KC1, 0.25 g NH4NO3, 0.01 mg FeSO4)
supplemented with 0.1% N-acetylglucosamine and 10%
sterilized insect cuticle (mole cricket, Scapteriscus abbrevia-
tus). Cultures were inoculated with 105 conidia/ml and
grown with aeration for 6 d at 25 C. Fungal cells were
lysed by grinding in liquid nitrogen and total RNA was
extracted using RNAWiz (Ambion). cDNA libraries were
constructed using the SMART RACE cDNA Amplification
kit (Clontech, CA) according to manufacturer instruc-
tions. For construction of E. coli expression plasmids, an
NdeI restriction site was incorporated into the forward
primer and an EcoRI site into the reverse primer. PCR
products were cloned directly into TOPO 2.1 using TOPO
TA cloning system and transformed into TOPO Top 10 E.
coli cells (Invitrogen, Carlsbad, CA). The TOPO 2.1 con-
structs were used for subcloning into the NdeI-EcoRI sites
of pET43.1a (Novagen, Darmstadt, Germany) for expres-



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Clinical and Molecular Allergy 2006, 4:12


sion using E. coli BL21 host strain harboring the pRARE
plasmid.

Protein expression, Western and immunoblotting
Overnight cultures of coli BL21 harboring pRARE along
with each respective pET43.1a based construct were
grown in 3 ml of LB (supplemented with 50 lg/ml ampi-
cillin and 12 ig/ml chloramphenicol) at 370C with aera-
tion. Fresh media (5-10 ml) was inoculated with aliquots
(0.1-0.2 ml) of the overnight culture, and samples were
incubated at 370C with aeration to an OD00 = 0.6-0.8.
T7 polymerase based expression of the recombinant pro-
teins was initiated by the addition of 1-1.5 mM (final
concentration) isopropyl-P-D-thiogalactopyranoside
(IPTG), and cultures were returned to the incubator for an
additional 2-3 hours. For extract preparation, cells were
harvested by centrifugation (10,000 x g, 10 min) and the
resultant pellet resuspended in 0.5 volumes TE (40 mM
Tris, 1 mM EDTA, 0.01% phenylmethylsulfonyl fluoride
(PMSF)). Cells were lysed by sonication (3 x 30 sec) on
ice, after which samples were centrifuged (10,000 x g, 10
min) and separated into soluble and pellet (containing
potential inclusion bodies) fractions. Samples of the
crude soluble and pellet extracts were denatured with 4x
LDS loading dye (Invitrogen) and boiled for 1-5 min
prior to separation by SDS-Polyacrylamide gel electro-
phoresis (PAGE) using the Invitrogen NUPage-MOPS
buffer system (10-12% Bis-tris polyacrylamide gels)
according to the manufacture's recommended protocols.
Gels were stained with Coomasie Blue R250 followed by
destaining with 10% methanol, 10% acetic acid solution.
For Western blots and immunodetection, samples were
analyzed by SDS-PAGE as described above, followed by
electroblotting to polyvinylidene-fluoride (PVDF) mem-
branes (Invitrogen). After blocking (TBST; 25 mM Tris-
HC1 buffer saline containing 0.1% Tween-20 and 10% dry
fat free milk), membrane were probed with either individ-
ual or pooled human sera as the primary antibody solu-
tion. Typically, sera were diluted in blocking buffer and
incubated with membranes overnight at 4-8 C with gen-
tle agitation. Membranes were washed 3 x using 50 ml
TBST for 15 min each. Binding of human IgEs was visual-
ized using a horseradish peroxidase (HRP) conjugated
goat anti-human IgE polyclonall) secondary antibody
(BioSource International, CA). Membranes were incu-
bated in secondary antibody (diluted 1:10,000 in block-
ing buffer) for 1 hr at room temperature, with gentle
agitation. After secondary antibody incubation mem-
branes were washed 3 times using 50 ml TBST and bands
visualized using the Immuno-Star HRP detection system
(Bio-Rad, Hercules, CA). Total protein membrane stain-
ing was performed using Ponceau S (Sigma, St. Louis,
MO).


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Analysis programs
Nucleotide manipulations and phylogenetic analyses
were performed using multiple software programs. Initial
sequence alignments were performed with ClustalW [29].
Alignment files (in Nexus format) were transferred to
Splitstree for analysis and construction of phylograms,
with typical bootstrap parameters set to 1000 [30].

Genbank submission
The isolated cDNA and genomic sequences of the four B.
bassiana genes have been submitted to Genbank with the
following accession numbers; Bb-Enol, DO767719; Bb-
f2, DO767720; Bb-Ald, DO767722; and Bb-Hex,
D0767722.

Results
Molecular characterization of four putative B. bassiana
allergens
EST (Expressed sequence tag) panning and screening of a
suppressive subtractive library (SSH) identified gene frag-
ments of four potential allergens by sequence homology.
The B. bassiana genes were designated as follows: Bb-
Enol, similar to Cladosporium herbarum enolase Cla h 6
[18]; Bb-f2, similar to Aspergillus fumigatus major allergen
Asp f2 [31]; Bb-Ald, similar to C. herbarum allergen Cla h
3, an aldehyde dehydrogenase [18]; and Bb-Hex, with
similarity to numerous fungal N-acetylhexosaminidases,
including the Penicillium chrysogenum Pen ch 20 allergen
[32].

Since the nucleotide fragments (200-300 bp) represented
only a portion of the entire gene sequence coding for each
protein, full length sequences were obtained by 5' and 3'
RACE PCR as needed. These results were used to assemble
the full length cDNA nucleotide sequences of the four
genes. Separate sets of primers were then designed for
amplification of the genomics DNA sequences of the
genes and for cloning into the E. coli pET43a-based pro-
tein expression system as described in the Methods sec-
tion. The lengths of the cloned cDNA and genomic
sequences, the number of introns, along with an analysis
of the predicted ORFs, detailing the number of amino
acids, molecular mass, and pIs of the deduced B. bassiana
proteins are given in Table 1. Top BLASTX search results
for each protein are also presented (Table 2).

The genomic sequence of Bb-Enol consisted of 1548 bp
from the start site to the stop codon and contained four
introns. The lengths of the introns were between 52-69
bp and were located in the first half of the gene. The cDNA
sequence of the open reading frame of Bb-Eno 1 consisted
of 1317 bp, constituting a protein of 438 amino acids
with a calculated molecular mass ~47 kDa. BLASTX simi-
larity searches of the complete Bb-Enol amino acid
sequence against the NCBI protein database confirmed


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Table I: Characteristics of the cloned B. bassiana genes and their predicted protein products


Protein ID putative function


Bb-Enol
Bb-f2
Bb-Ald
Bb-Hex


genomic clone (bp) # of introns cDNA clone (bp) # of amino acids Molecular mass (KDa) pl (protein)


Enolase
Unknown
aldehyde- dehyrogenase
hexosaminidase


the initial observation, resulting in high similarity to eno-
lases derived from numerous fungal species, including A.
fumigatus, Penicillium citrinum, Alternaria alternate, and C.
herbarum.

The genomic sequence of Bb-f2 consisted of 845 bp (start
to stop codon) and contained one intron that began at bp
412 and was 59 bp in length. The coding sequence of Bb-
F2 consisted of 261 amino acids, with a calculated molec-
ular mass of 28 kDa. BLASTX similarity searches con-
firmed that Bb-f2 displayed high sequence similarity to
the A. fumigatus major allergen Asp f 2.

The Bb-Ald genomic clone contained two introns; the first
106 bp in length, 62 bp from the ATG start codon, and the
second, 59 bp in length, starting 568 bp from the start
codon. The total size of the genomic clone was 1659 bp
(start to stop codon), with the cDNA sequence consisting
of 1494 bp coding for a proteins comprised of 497 amino
acids with a calculated molecular mass of 53 kDa. BLASTX


similarity searches using the complete Bb-Ald sequence as
the query revealed similarity to aldehyde dehydrogenases,
including those from A. alternate and C. herbarum.

The genomic clone corresponding to Bb-Hex was 1959 bp
in length (start to stop codon) and did not contain any
introns. The open reading frame coded for a protein con-
sisting of 652 amino acids with a calculated molecular
mass of 72 kDa. BLASTX similarity searches confirmed
high sequence similarity to fungal N-acetylhexosamini-
dases.

Expression of recombinant B. bassiana proteins
The coding sequences of the four B. bassiana genes were
subcloned into the pET43.1a expression vector as
described in the Methods. The integrity of all clones was
verified by sequencing of the inserts. The recombinant B.
bassiana proteins were expressed in E. coli strain BL21 har-
boring the pRARE plasmid that contains the genes for the
expression of rare tRNAs (Fig. 1, initial experiments using


Table 2: BLASTX search results using full-length B. bassiana sequences


Search Results


Organism


Function


Alternaria alternate
Cladosponum herbarum
Aspergillus fumigatus
Neurospora crassa
Penicillium citrinum

Aspergillus fumigatus
Aspergillus nidulans
Candida albicans
Candida albicans

Alternaria alternate
Cladosponum herbarum
Cladosporium fulvum
Neurospora crassa
Aspergillus nidulans

Metarhizium anisopliae
Aspergillus fumigatus
Aspergillus oryzae
Penicillium chrysogenum


Allergen I.D. Accession number


enolase
enolase
enolase
enolase
enolase

major allergen
antigen I
pH regulated antigen
fibrinogen binding mannoprotein

aldehyde dehydrogenase
aldehyde dehydrogenase
aldehyde dehydrogenase
aldehyde dehydrogenase
aldehyde dehydrogenase

N-acetylhexosaminidase
N-acetylhexosaminidase
N-acetylhexosaminidase
N-acetylhexosaminidase


Alt a 6
Cla h 6
Asp f 22w
-1
Pen c 22w

Asp f2





Alta 10
Cla h 3








Pen ch 20


D0767719
U82437
X78226
AF284645
XM323150
AF254643
D0767720
AAC69357
XP659435
AAC00525
AAC49898
DO767721
X78227
X78228
AF275347
XM951769
XM653066
D0767722
D0000319
XM742214
AB085840
AAB34785


E-value


<10-100oo
<10-10oo
<10-100oo
<10-10oo
<10-10oo

10-64
10-55
10-52
10-52

<10-1oo
<10-10oo
<10-1oo
<10-1oo
<10-1oo

<10-1oo
<10-1oo
<10-1oo
10-47


I Dash indicates that it is unknown whether the protein is an allergen.


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Ouery


Bb-Enol






Bb-f2





Bb-Ald






Bb-Hex


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.- --


MW
(kDa)
191- m

97-

64- -O


1234


5678


S -


MW
(kDa)
97-
64-
51-

39-i

28-

19-

14-1


[


-w u


39- .


a -


a0W MW -,.- -


Figure I
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 indi-
cated expression plasmid constructs; lanes I) and 2);
pET41a:Bb-Enol, lanes 3) and 4) pET41a:Bb-f2, lanes 5) and
6) pET41a:Bb-Ald, lanes 7) and 8) pET41a:Bb-Hex. Unin-
duced cell cultures, lanes 1), 3), 5), and 7). IPTG induced cell
cultures, lanes 2), 4), 6), and 8).





a BL21 strain lacking the pRARE plasmid resulted in little
to no expression). Fractionation of the crude extracts into
soluble and insoluble (presumably inclusion bodies) frac-
tions revealed the B. bassiana proteins to be largely in the
insoluble fraction (Fig. 2). In some instances, induction of
the pET:Bb-Enol clone by IPTG resulted in the production
of two bands, the first having the expected mass of 47 kDa
and a second smaller band with a mass ;45 kDa (Fig 1,
lane 2). Similarly, the Bb-F2 clone also appeared to pro-
duce two protein bands of 28 kDa (Figure 1, lane 4). Fur-
ther experimentation revealed that these bands were due
to cleavage during heat denatuation (Fig 3).

IgE immunoblot analysis of recombinant proteins
Immunoblots were used in order to determine whether
human IgEs could bind the recombinant B. bassiana pro-
teins. Crude E. coli extracts containing the expressed pro-
teins were resolved by SDS-PAGE and transferred to PVDF
membranes as described in the Methods. Initial experi-
ments were performed using blots containing the four
expressed proteins as well as a crude B. bassiana extract
(positive control), that were probed with one of two sera
pools containing serum from ten patients each, pools A-J


28-4 -

19- L I



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 I), 3), 5) and 7), and pellet fractions, lanes 2), 4), 6), and
8). Expression of Bb-Eno I, lanes I) and 2), Bb-f2, lanes 3) and
4), Bb-Ald, lanes 5) and 6), and Bb-Hex, lanes 7) and 8).


and K-T (Fig. 4). Each blot was treated with 0.2 ml of each
serum (1:35 dilution, final concentration). The blot
probed with pool A-J revealed strong IgE binding of the
two protein bands corresponding to BbEnol, as well as
several reactive (background) E. coli bands. The B. bassiana
crude extract reacted with a variety of IgEs present in the
sera as has been previously reported. From the sera tested,
faint IgE binding to Bb-Ald was noted, with no visible IgE
binding observed for Bb-f2 and Bb-Hex. Control blots
using E. coli crude extracts derived from cells harboring
the vector with no insert resulted in essentially the same
background bands as seen with extracts containing the
expressed proteins.

In order to confirm the binding of IgEs to Bb-Ald, addi-
tional experiments using smaller sera pools and higher
final concentrations of individual sera were performed.
Five sera pools, each containing 1:5 dilutions of two sera,
and designated as AB, CD, EF, GH, IJ, KL, MN, OP, QR,
and ST were created. In one set of experiments pools AB,
CD, EF, GH, and IJ were then used to probe membranes
containing Bb-Enol, Bb-f2, and Bb-Ald, (Fig. 5, Bb-Hex
was omitted due to the lack of reactivity in preliminary


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1 2 3 4 567


51- 4-


p2-


I
a-


Clinical and Molecular Allergy 2006, 4:12












(b) (c)
1 2 3 4 1 2 3 4


64-
51- m
39- ..

28- -


W


14-





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; I min heat dena-
turation at 95C, panel A), 5 min, 95C, panel B), and 20 min,
95C, panel C), Lanes correspond to crude extracts contain-
ing, lane I) Bb-Eno lane 2) Bb-f2, lane 3) Bb-Ald, and lane 4)
Bb-Hex.



experiments). These results confirmed IgE binding to Bb-
Enol (strong signal from pools AB and EF, with weaker
signal from pool GH) and to Bb-Ald (pools AB and GH).
Not too surprisingly, IgE binding of "background bands",
i.e. antigens derived from the E. coli extracts were highly
variable between pools. Using these sera pools, no IgE
binding was observed to Bb-f2. In a second series of exper-
iments the pools were used to probe membrane strips
containing only Bb-Ald extracts. IgE binding of Bb-Ald
was noted using pools AB, GH, OP, and ST (Fig. 6). Since
pool AB resulted in strong signals to both Bb-Enol and
Bb-Ald, further experiments were performed using the
individual sera (either A or B) to probe membranes con-
taining all four B. bassiana recombinant proteins (Fig 7).
These results revealed that serum A contained IgEs that
bound to Bb-Enol and Bb-Ald, whereas serum B con-
tained IgEs reactive only to Bb-Enol.

Phylogenetic analyses
Bb-Enol displayed high sequence similarity to fungal
enolases several of which are known allergens. A phylo-
gram was constructed using the amino acid sequences of
21 fungal enolases as well as those of Drosophila mela-
nogaster, E. coli, and the rubber plant, Hevea brasiliensis, a
known potent allergen (Fig. 8). Of the enolases examined,
nine have been identified as allergens (designated with an
asterisk in the figure). These proteins do not appear to
cluster in any discernable pattern and are equally distrib-
uted throughout the phylogram. Similarly, an analysis of
the available fungal aldehyde dehydrogenases failed to


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MW (a)
(kDa) 1 2 3
191-


L


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reveal any discernable pattern or clustering of the known
allergens.

Discussion
Allergy is a hypersensitive response of the immune system
and fungi are important triggers of respiratory and other
forms of allergies [5,33-35]. As alternatives to chemical
pesticides, entomopathogenic fungi such as Metarhizium
anisopliae and Beauveria bassiana hold promise as biologi-
cal control agents, and both organisms have been EPA
approved for commercial control of a variety of arthropod
pests [8-11]. The process of fungal infection of insect tar-
gets involves the use of infectious propagules, typically
conidiaa) spores, which attach and germinate across host
surfaces. Growing fungal cells then begin to penetrate the
cuticle and proliferate within the insect body, ultimately
resulting in the death of the host [6,36,37]. Use of these
biological pesticides, however, is likely to lead to the dis-
persal of inhalable fungal particles. Several studies have
demonstrated the potential of these fungi in eliciting aller-
gic reactions. [12,14,38]. Some occupational allergy to M.
anisopliae has been noted and immune and pulmonary
responses characteristic of allergy were observed in Balb/c
mice challenged with M. anisopliae extracts [39,40]. Fur-
thermore, allergen-triggered airway hyperresponsiveness
and lung pathology occurred in mice sensitized with this
fungus [41]. The allergenic potential of B. bassiana has
been confirmed by intradermal skin testing, and numer-
ous IgE reactive proteins, some of which are cross-reactive
among allergens from other fungi have been noted in this
organism [14]. To date, however, there have been no
reports detailing the molecular identification of B. bassi-
ana (or M. anisopliae) IgE-reactive antigens.

The present study describes the cloning and expression of
four putative B. bassiana allergens and demonstrated IgE-
reactivity for two of the recombinant proteins using sera
derived from patients displaying mold allergies. Bb-Enol
has a calculated monomer molecular mass of 47.4 kDa
and displays similarity to enolases that form an exten-
sively studied group of allergens. IgE cross reactivity
between the enolases of C. herbarum, A. alternate, C. albi-
cans, and A. fumigatus has been well characterized and it is
likely that the B. bassiana protein would also be recog-
nized by the same IgEs. Modeling of the C herbarum eno-
lase using the solved crystal structure of the S. cerevisiae
enolase was used to construct 10 recombinant peptides
spanning the length of the C. herbarum enolase [42]. Six of
these peptides, distributed throughout the entire length of
the protein showed IgE-binding activity. One of the pep-
tides encompassed a region that overlapped with the
other 5 IgE reactive peptides and formed, based upon the
modeling, an extended structure that twice spanned the
body of the globular protein and reached the surface three
times. This sequence was therefore deemed contain at






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MW
(kDa)

191-


97-

64-

51-


39-



28-


A-J
1 2 3 4 5


K-T
1 2 3 4


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 I) Bb-Enol, 2) Bb-f2,. 3) Bb-Ald, 4) Bb-Hex. Lane 5) 40 pig crude B. bassiana extract.


least one immunodominant IgE epitope, and sequence
analyses revealed a highly similar stretch of amino acids in
the deduced B. bassiana enolase sequence. Approximately
20% of the sera tested (4-6/20) displayed positive IgE
reactivity to the recombinant Bb-Eno 1, indicating that this
protein is likely to be a significant allergen in B. bassiana.

Bb-Ald was similar to the A. alternate Alt a 10 and C. her-
barum Cla h 3 proteins, both of which have been charac-
terized as aldehyde dehydrogenases [18]. In a survey of
allergens recognized by patients with mold allergies, 2%
displayed IgE reactivity to Alt a 10, whereas 36% displayed
reactivity to Cla h 3 [18]. Based upon these results, Cla h
3 was classified as an important allergen and Alt a 10 as a
minor allergen. Only one (out of twenty) of our sera dis-
played strong reactivity to Bb-Ald, indicating that this pro-
tein is indeed an allergen, however due to our small


sample size, it is not possible to draw any definitive con-
clusions regarding the importance of this protein as a B.
bassiana allergen.

Bb-f2 showed sequence homologies to the A. fumigatus
Asp f2 major allergen and the fibrinogen binding protein
from C. albicans [43]. In A. fumigatus, Asp f2 appears to be
expressed as a 55-kDa mycelial glycoprotein as well as a
37-kDa culture filtrate presumably deglycosylated protein
(the calculated molecular mass of Asp f2 is 29 kDa, the
reason for the discrepancy is unclear but may be attributed
to specific C-terminal amino acid residues), both of which
are IgE reactive [31]. Asp f2 also appears to interact with
extracellular matrix proteins such as laminin, and exhib-
ited IgE binding from sera derived from patients with
allergic bronchopulmonary aspergillosis (ABPA) and cyc-
tic fibrosis-ABPA patients, but not from sera isolated from


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Clinical and Molecular Allergy 2006, 4:12







http://www.clinicalmolecularallergy.com/content/4/1/12


CD
2 3 1 2


EF
3 1 2


GH
3 1


IJ
2 3 1


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 I) Bb-Enol, 2) Bb-f2,. 3) Bb-Ald.


(1) (A) (B)
12 3 4 1 2 3 4 12 3 4


a- I-


Figure 6
Immunoblot analysis of recombinant Bb-Ald. PVDF mem-
brane strips containing crude extracts of E. coli expressed Bb-
Ald were probed with I 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 concen-
tration 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 fol-
lows, lane I) Bb-Enol, 2) Bb-f2,. 3) Bb-Ald, and 4) Bb-Hex.
Panel I) represents Ponceau S staining of the PVDF mem-
brane after transfer.


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AB
1


MW
(kDa)

97-

64-
51-

39-


28-


19-


14-


2 3


AB CD GH IJKL MN OP QR ST





aW OR


MW
(kDa)
97-
64-
51-

39-

28-

14-


--- fl.


Clinical and Molecular Allergy 2006, 4:12








Clinical and Molecular Allergy 2006, 4:12


1 00 N frontahs
100
100 D melanogaster
1 00 H brasaensis*
100 C herbarum*
1 00 A alteata*

1 00
100 C unata
1 A00 oryzae
"A fumtgatus*
1 00 0 991 00A dulan
-00 P chrysogenum
P P ctannum*
S98
100
0 0- N crassa


1 00
0 rubra*
099
100 C albicans*
00 K lachs
100 A gossypu
100
C glabata
oo S ceremsae*
100
0--0 D hansenm
100 S pombe
100
-100 -E coh
100
1-00- C elegant


Figure 8
Full length amino acid sequences of 24 enolases deposited in
the NCBI Genbank database were used to construct an eno-
lase 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.



A. fumigatus-sensitized allergic asthma (and normal con-
trol subjects) [31]. Thus, the observed lack of IgE reactivity
to Bb-f2 may be attributed to the lack of ABPA patients in
our sera samples.

The original cDNA fragment corresponding to Bb-Hex dis-
played the highest similarity to the N-acetylhexosamini-
dase of P. chrysogenum (Pen ch 20), that has been
identified as an allergen [32]. Subsequent, full length
cDNA cloning and characterization resulted in higher
similarity to other fungal hexosaminidases that have not
been characterized as allergens (see E-values in Table 2).
None of our sera samples displayed IgE reactivity to
recombinant Bb-Hex, however, due to our low sample
size the possibility cannot be excluded that this protein
represents a B. bassiana allergen.


http://www.clinicalmolecularallergy.com/content/4/1/12




Our results confirm the potential allergenicity of B. bassi-
ana by the molecular and immunological characterization
of specific allergens from this organism and suggest that
some precautions should be taken into account in biolog-
ical control applications using entomopathogenic fungi.
It is, however, important not to overstate the potential
risks (versus benefits) and the overall safety with respect
to allergenicity of these fungi may be similar to that of
baker's yeast from which allergens, including enolase,
have also been isolated [44,45]. While it is not possible to
determine the fraction of the total allergen production the
four isolated proteins represents, immunoblot compari-
sons between the isolated proteins and the reactivity of
crude B. bassiana extracts indicated the presence of numer-
ous additional allergens that have yet to be characterized,
some of which may represent more highly antigenic
epitopes. The isolation of putative B. bassiana allergens
described in this report relied upon identifying molecules
by the resemblance of their DNA sequences to previously
identified allergens and future experiments using alter-
nate approaches (e.g. phage display [46]) may be needed
to identify additional allergens. Finally, although much
work has been performed in regards to isolating and char-
acterizing fungal allergens, the roles of these proteins in
fungal processes such as development and pathogenesis
remains obscure. As a genetically amenable organism and
a pathogen of arthropods, B. bassiana represents a novel
system to examine the relationship between allergenicity
and (insect) pathogenesis. Targeted gene-knockouts, for
instance, can be used to probe affects upon virulence and
interactions between specific allergen and arthropod
innate immune systems.

Conclusion
Cloning, sequencing, and heterologous expression of four
putative B. bassiana allergens was performed. Recom-
binant proteins corresponding to Bb-Enol and Bb-Ald,
but not Bb-f2 and Bb-Hex, displayed IgEs reactivity
against sera from patients with mold allergies. Due to the
low sera sample numbers used, it cannot be excluded that
Bb-f2 and Bb-Hex are allergens, and further testing is war-
ranted. Bb-Enol was similar to enolases that represent a
well characterized group of major allergens. Bb-Ald was
similar to aldehyde dehydrogenases that are considered
major allergens in some fungal species, but minor aller-
gens in others. The molecular identification ofB. bassiana
allergens can lead diagnostic methods for determining
sensitization to this organism and provides a rational
basis for allergen attenuation in order to yield safer bio-
logical control products. The B. bassiana-arthropod inter-
action may represent a novel model system to examine
the relationships between allergenicity and pathogenicity.


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Clinical and Molecular Allergy 2006, 4:12


Abbreviations
ABPA, allergic bronchopulmonary aspergillosis, BLAST,
basic local alignment search tool, EPA, Environmental
Protection Agency, E-value, expect score, EDTA, ethylene-
diaminetetraacetic acid, EST, expressed sequence tags,
HRP, horseradish peroxidase, IgE, immunoglobulin E,
IPTG, isopropyl-b-D-thiogalactoside, LB, Luria-Bertani
broth, NCBI, National Center for Biotechnology Informa-
tion, PAGE, polyacrylamide gel electrophoresis, PCR,
polymerase chain reaction, PMSF, phenylmethyl sulfonyl
fluoride, PVDF, polyvinylidene fluoride, RACE, rapid
amplification of cDNA ends, SDS, sodium duodecyl sul-
fate, SSH, suppressive subtractive hybridization, TBS, Tris
buffered saline.


Competing interests
The authors) declare that they have no competing inter-
ests.


Authors' contributions
GSW carried out the molecular, immunological, and
other in vitro experiments, and participated in the design
of the study. SWH participated in the design of the study
and provided technical support for the project. NOK con-
ceived of the study, participated in its design and coordi-
nation, and drafted the manuscript.

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