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Swee Yang Low1, Karen Dannemiller1, Maosheng Yao2, Naomichi Yamamoto1, and Jordan Peccia1
1Yale University, New Haven, CT, USA 2Peking University, Beijing, China
Growth temperature strongly influence the allergenicity of Aspergillus fumigatus
spores
NIEHS RES015312A
• Not a well researched subject- as opposed to microbiological content in soils and water l very dilute concentrations
l poor cross-training between aerosol scientists and biologists
l historical bias against the airborne route of infection
Microbial Material in the Air
Measurement of allergens
●Allergenic disease is a major cause of illness and disability in the US. Up to 40% of the members of the general public have developed IgE antibodies against environmental antigens (Pope et al., Indoor Allergens 1993).
● >700 different biological allergens. (I.U.I.S., The official 'List of Allergens'. 2011).
●Type 1 allergenic disease is increasing (Ring, J., et al., Current Opinion
in Immunology, 2001. 13: p. 701).
Peccia, Milton, Reponen, and Hill. Environmental Science and Technology, 2008
Allergenic fungal diversity through 454 pyrosequencing
Epicoccum
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ambiguous classifications
non-allergenic fungi
allergenic fungi
Cladosporium
Epicoccum
Cryptococcus
Pleospora Alternaria Penicillin Aspergillus
Rhodotorula Stachybotrys Stemphylium
total fungal sequences
alle
rgen
frac
tion
MAST cells and BASOPHILS with attached IgE releases histamine or other inflammatory agents upon binding with allergenic epitope
Complexity of biological allergens
MAST CELLS/BASOPHILS
IgE
Asp f 1
Allergenic spores have multiple IgE binding sites
Aspergillus fumigatus has 23 allergenic proteins. (proteins have diversity of functions e.g. ribotoxin, heat shock protein) IgE can bind to multiple sites within a specific protein
TTGGSSSTPHGKDDHYLLEFFWTCINGIVAHQR
1
3
Hypothesis and Objectives
Objective 1: Determine if the allergen potency (IgE binding capacity) of A. fumigatus spores is a function of temperature.
HYPOTHESIS: Allergen potency is a function of the environment in which a spore is produced
Objective 2: Track the expression of allergen encoding genes in fungal spores produced at variable temperatures.
• Allow Aspergillus fumigatus conidia to form at temperatures ranging from 14°C to 32°C and determine the relative allergenicity per spore.
Approach
Figure 1. Allergenicity per spore is defined as the ability to inhibit the binding of human IgE from sensitize patients to a immobilized Aspergillus fumigatus allergen extract.
• Quantify the transcription levels of allergen encoding genes in spores grown at 17°C to 32°C to determine if the variation in allergenicity has a transcriptional basis.
Figure 2. Gene expression microarrays were use to compare global and allergen encoding gene transcription in A. fumigatus that sporulated at 17°C and 32°C.
high allergenicity low allergenicity
Bind A. fumigatus spores with IgE from sensitized patients
Unbound IgE is mixed with immobilized A. fumigatus extract
17°C
32°C
Sporulate at different temperatures
Extract mRNA
Label cDNA with Cy5 & Cy3
• Allow Aspergillus fumigatus conidia to form at temperatures ranging from 14°C to 32°C and determine the relative allergenicity per spore.
Approach
Figure 1. Allergenicity per spore is defined as the ability to inhibit the binding of human IgE from sensitize patients to a immobilized Aspergillus fumigatus allergen extract.
• Quantify the transcription levels of allergen encoding genes in spores grown at 17°C to 32°C to determine if the variation in allergenicity has a transcriptional basis.
Figure 2. Gene expression microarrays were use to compare global and allergen encoding gene transcription in A. fumigatus that sporulated at 17°C and 32°C.
high allergenicity low allergenicity
Bind A. fumigatus spores with IgE from sensitized patients
Unbound IgE is mixed with immobilized A. fumigatus extract
17°C
32°C
Sporulate at different temperatures
Extract mRNA
Label cDNA with Cy5 & Cy3
• Allow Aspergillus fumigatus conidia to form at temperatures ranging from 14°C to 32°C and determine the relative allergenicity per spore.
Approach
Figure 1. Allergenicity per spore is defined as the ability to inhibit the binding of human IgE from sensitize patients to a immobilized Aspergillus fumigatus allergen extract.
• Quantify the transcription levels of allergen encoding genes in spores grown at 17°C to 32°C to determine if the variation in allergenicity has a transcriptional basis.
Figure 2. Gene expression microarrays were use to compare global and allergen encoding gene transcription in A. fumigatus that sporulated at 17°C and 32°C.
high allergenicity low allergenicity
Bind A. fumigatus spores with IgE from sensitized patients
Unbound IgE is mixed with immobilized A. fumigatus extract
17°C
32°C
Sporulate at different temperatures
Extract mRNA
Label cDNA with Cy5 & Cy3
Human IgE binding assay for allergenicty
Characteristic dose-response curve,
IgE bind to A. fumigatus proteins
-50 0
50 100 150 200 250 300 350 400 450
1.0E+05 1.0E+06 1.0E+07
Rel
ativ
e A
llerg
enic
ity
Spore Concentration (spores/ml)
105 106 107
17 oC
(P < 0.01, ANOVA)
Effects of temperature on allergen potency
Effects of temperature on culturability of A. fumigatus
(P < 0.005, t test)
Effects of temperature on allergen potency
• Allow Aspergillus fumigatus conidia to form at temperatures ranging from 14°C to 32°C and determine the relative allergenicity per spore.
Approach
Figure 1. Allergenicity per spore is defined as the ability to inhibit the binding of human IgE from sensitize patients to a immobilized Aspergillus fumigatus allergen extract.
• Quantify the transcription levels of allergen encoding genes in spores grown at 17°C to 32°C to determine if the variation in allergenicity has a transcriptional basis.
Figure 2. Gene expression microarrays were use to compare global and allergen encoding gene transcription in A. fumigatus that sporulated at 17°C and 32°C.
high allergenicity low allergenicity
Bind A. fumigatus spores with IgE from sensitized patients
Unbound IgE is mixed with immobilized A. fumigatus extract
17°C
32°C
Sporulate at different temperatures
Extract mRNA
Label cDNA with Cy5 & Cy3
• Allow Aspergillus fumigatus conidia to form at temperatures ranging from 14°C to 32°C and determine the relative allergenicity per spore.
Approach
Figure 1. Allergenicity per spore is defined as the ability to inhibit the binding of human IgE from sensitize patients to a immobilized Aspergillus fumigatus allergen extract.
• Quantify the transcription levels of allergen encoding genes in spores grown at 17°C to 32°C to determine if the variation in allergenicity has a transcriptional basis.
Figure 2. Gene expression microarrays were use to compare global and allergen encoding gene transcription in A. fumigatus that sporulated at 17°C and 32°C.
high allergenicity low allergenicity
Bind A. fumigatus spores with IgE from sensitized patients
Unbound IgE is mixed with immobilized A. fumigatus extract
17°C
32°C
Sporulate at different temperatures
Extract mRNA
Label cDNA with Cy5 & Cy3
• Allow Aspergillus fumigatus conidia to form at temperatures ranging from 14°C to 32°C and determine the relative allergenicity per spore.
Approach
Figure 1. Allergenicity per spore is defined as the ability to inhibit the binding of human IgE from sensitize patients to a immobilized Aspergillus fumigatus allergen extract.
• Quantify the transcription levels of allergen encoding genes in spores grown at 17°C to 32°C to determine if the variation in allergenicity has a transcriptional basis.
Figure 2. Gene expression microarrays were use to compare global and allergen encoding gene transcription in A. fumigatus that sporulated at 17°C and 32°C.
high allergenicity low allergenicity
Bind A. fumigatus spores with IgE from sensitized patients
Unbound IgE is mixed with immobilized A. fumigatus extract
17°C
32°C
Sporulate at different temperatures
Extract mRNA
Label cDNA with Cy5 & Cy3
Gene expression microarrays of A. fumigatus sporulating at 17oC and 32oC
Global expression pattern – protein production upregulated at lower temperatures
Expression of allergen encoding genes
Investigations in progress:
Low oxidant levels increase the production of peroxisomal membrane protein (Asp f3). Homologs of the genes encoding for the PMP are found in 23 or 25 allergenic fungi in with genomes have been sequenced.
●How do indoor and atmospheric pollutants impact the allergenicity of different fungal allergens?
NO2
O3
NOx
(1)
(2)
Conclusions
● Environmental conditions during sporulation influence the allergenicity “IgE binding capacity” and allergen production in A. fumigatus spores.
Human IgE binding
Total and allergenic protein
Microarray and qPCR gene expression
● Culturability and qPCR can underestimate (5 to 50 times) the allergenicity of A. fumigatus spores that were produced at lower temperature.