Impact of pAA plasmid- and chromosome-encoded factors on the high virulence of EHEC O104:H4

Basic data for this project

Description

Enterohemorrhagic Escherichia coli (EHEC) can cause severe foodborne illness in humans, which is typically characterized by bloody diarrhea and can progress to hemolytic uremic syndrome (HUS) in up to 10% of the cases. EHEC of serotype O104:H4 (EHEC O104:H4) was identified as the causative agent of the largest German outbreak (May-July 2011) during which 3842 people were infected, and of these 855 (22%) developed HUS. Besides having a chromosomally integrated Shiga toxin 2 encoding bacteriophage, EHEC O104:H4 carries a pAA plasmid encoding the aggregative adherence fimbriae I cluster (AAF/I; a characteristic virulence feature of enteroaggregative E. coli) mediating the tight adherence of the outbreak strain to cultured human epithelial cells. The main objective of our project is to characterize factors and mechanisms contributing to the high virulence of EHEC O104:H4. We plan to investigate known, as well as to discover novel virulence-associated traits using a transcriptomics approach based on differential RNA sequencing (dRNA-seq), a recently established method for the high throughput analysis of primary transcriptomes. In the first part of our two thematically linked project parts we will investigate the impact of the pAA plasmid on EHEC O104:H4 virulence. The importance of pAA in disease severity was recently demonstrated by the observation that the outbreak strain can lose pAA during the course of illness and that this loss is correlated with a significantly reduced progression to HUS. Specifically, we will analyze the pAA gene regulation with focus on known virulence-associated genes. Moreover, we hypothesize that both the pAA plasmid itself and the interplay between pAA- and chromosome-encoded factors of EHEC O104:H4 are important determinants for the high virulence. Therefore, we will determine the pAA impact on bacterial growth, survival and biofilm formation, as well as on the host-pathogen interaction. Furthermore, we will investigate pAA-dependent chromosomal gene regulation by comparative dRNA-seq analysis of the pAA-positive and negative EHEC O104:H4.In the second part we aim to determine previously unknown players contributing to EHEC O104:H4 high virulence. Strain-specific virulence genes (e.g. stx2, AAF/I cluster) are already recognized as important determinants for the EHEC O104:H4 virulence; however, we hypothesize that a strain-specific regulation of common E. coli genome features also significantly contributes to the exceptional virulence of EHEC O104:H4. Therefore, we will perform comparative dRNA-seq to identify such regions, which are present in EHEC O104:H4 and in other less pathogenic or commensal strains but are differentially transcribed. Furthermore, we will perform functional studies to evaluate the impact of the discovered quantitative and qualitative transcriptome differences on the high virulent phenotype of EHEC O104:H4.