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

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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.