Text of Growth temperature strongly influence the allergenicity of Aspergillus fumigatus spores
1. Growth temperature strongly influence theallergenicity of Aspergillus fumigatus sporesSwee Yang Low1, Karen Dannemiller1, MaoshengYao2, Naomichi Yamamoto1, and Jordan Peccia11YaleUniversity, New Haven, CT, USA2Peking University, Beijing, ChinaNIEHS RES015312A
2. Microbial Material in the Air 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 andbiologists l historical bias against the airborne route of infection 3. 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).Type 1 allergenic disease isincreasing (Ring, J., et al., Current Opinion in Immunology, 2001. 13: p. 701).Peccia, Milton, Reponen, and Hill. Environmental Scienceand Technology, 2008 >700 different biological allergens. (I.U.I.S., The official List of Allergens.2011). 4. Allergenic fungal diversity through454 pyrosequencing Stemphylium$!!"# StachybotrysRhodotorulaAspergillus total fungal ,!"# PenicillinAlternariasequences +!"# Pleosporaallergen fraction *!"# Cryptococcusambiguous )!"# Epicoccumallergenic classificationsfungi (!"#non-allergenic !"# fungi &!"# CladosporiumEpicoccum %!"# $!"#!"# 5. Complexity of biological allergensIgE MAST CELLS/BASOPHILSMAST cells and BASOPHILS withattached IgE releases histamineor other inflammatory agentsupon binding with allergenicepitopeAsp f 1 6. Allergenic spores have multiple IgEbinding sitesAspergillus fumigatus has 23 allergenicproteins. (proteins have diversity offunctions e.g. ribotoxin, heat shock 3protein)IgE can bind to multiple sites within a 1specific proteinTTGGSSSTPHGKDDHYLLEFFWTCINGIVAHQR 7. Hypothesis and ObjectivesHYPOTHESIS: Allergen potency is a function of the environment inwhich a spore is producedObjective 1: Determine if the allergen potency (IgE binding capacity)of A. fumigatus spores is a function of temperature.Objective 2: Track the expression of allergen encoding genes infungal spores produced at variable temperatures. 8. Human IgE binding assay for allergenictyApproach Allow Aspergillus fumigatus conidia to form at temperatures rangi32C and determine the relative allergenicity per spore.FiBind A. fumigatus pespores with IgE fromthesensitized patients binfroai Unbound IgE is mixed fum with immobilized A.ex fumigatus extract high allergenicitylow allergenicity 9. Characteristic dose-response curve,IgE bind to A. fumigatus proteins45040017 oC350 Relative Allergenicity300250200150100 500-50 1.0E+051051.0E+061061.0E+07 107 Spore Concentration (spores/ml) 10. Effects of temperature on allergen potency (P < 0.01, ANOVA) 11. Effects of temperature on culturability of A.fumigatus (P < 0.005, t test) 12. spores with IgE fromsensitized patients binding of human IgE Gene expression microarrays of A. potency Effects of temperature on allergen fumigatus from sensitize patients toa immobilized Aspergillus sporulating at immobilizedmixed 32 C Unbound IgE is and with17oC A.ofumigatus allergenextract.fumigatus extracthigh allergenicitylow allergenicity Quantify the transcription levels of allergen encoding genes in spores grown at 17C to 32C todetermine if the variation in allergenicity has a transcriptional basis.17CFigure 2. Gene expressionmicroarrays were use toSporulate atcompare global and allergenExtractLabel cDNAdifferentmRNA with Cy5 & Cy3 encoding gene transcription intemperaturesA. fumigatus that sporulated at17C and 32C.32C 13. Global expression pattern proteinproduction upregulated at lower temperatures 14. Expression of allergen encoding genes 15. Investigations in progress:How do indoor and atmospheric pollutants impact theallergenicity of different fungal allergens? (1) O3 NOxNO2 (2) 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. 16. Conclusions Environmental conditions during sporulation influencethe allergenicity IgE binding capacity and allergenproduction in A. fumigatus spores.Human IgETotal and Microarray andbindingallergenicqPCR gene protein expression Culturabilityand qPCR can underestimate (5 to 50times) the allergenicity of A. fumigatus spores that wereproduced at lower temperature.