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Obersteiner, Andrea (2017): Pollen associated microbiome and its relationship to pollution and allergens. Dissertation, LMU München: Fakultät für Biologie
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Obersteiner_Andrea.pdf

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Abstract

In the past decades the prevalence of pollen-related respiratory allergies has increased rapidly. Causative agents are mostly airborne pollen grains of trees and grasses. The allergenic potential of these pollen grains depends on the amount of produced allergenic proteins (Bet v 1, Phl p 5) and non-allergenic adjuvant compounds, so called PALMs (pollen-associated lipid mediators). These compounds also play a role in the defense mechanisms of plants against biotic and abiotic stress. The first aim of this thesis was to analyze the composition of the microbial colonization of allergenic birch and timothy grass pollen using cultivation dependent and cultivation independent molecular methods. In addition, the microbes should be localized on the pollen grains. A second aim was to uncover the relationship between urbanization-related pollution parameter (nitrogen dioxide NO2, ozone O3, ammonia NH3, Urbanization Index UI), microbial α-diversity (Simpson, Shannon) colonizing the pollen and the expression of allergens and PALMs as well as stress induced NADPH-oxidase activity in the pollen. To reach these aims, universal media were inoculated with pollen suspensions to isolate microbes and identify them by phylogenetic analyses (arb, BLAST). Furthermore microbial patterns and diversity were determined using community DNA isolated from pollen of birch and timothy grass collected in three consecutive years at different locations using terminal restriction fragment length polymorphism (tRFLP) analysis. These microbial patterns were used for calculating Spearman-correlations with pollution and allergen parameters. 16S-rDNA amplicon sequencing (Illumina MiSeq) was further used to determine abundances on bacterial family and genus levels. Localization studies were performed via Fluorescent in situ Hybridization (FISH) and confocal Laser-scanning microscopy (CLSM). Most of the isolated microbes are known as environmental organisms from soil, plant and water. Several species show potential interactions with plants, but also possible pathogenicity against humans. Furthermore, a plant-species specific microbiome was characterized on pollen of birch and timothy grass as evidenced by tRFLP- and 16S rDNA amplicon sequencing. Accordingly, FISH- and CLSM-studies showed dominant groups of α-Proteobacteria on the surface of birch and γ-Proteobacteria on timothy grass pollen. Correlation analyses of microbial tRFLP-pattern (bacterial diversity) to pollution parameters and the expression of allergen-related compounds showed significant results for several parameters (NO2, O3, UI; Bet v 1, PALMs). The results demonstrate that bacterial diversity correlates with anthropogenic factors like pollution, which in turn might impact the expression of allergens and hence, alters the immune-inflammatory potential of pollen.