Investigations of the impact of different determinants on the virulence and fitness of EHEC O104:H4

肠出血性大肠杆菌(EHEC)可导致严重的食源性疾病，其典型特征是血性腹泻，并在约10%的病例中进展为溶血性尿毒症综合征(HUS)。EHEC O104：H4被确定为德国最大的疫情(2011年5月至7月)的病原体，在此期间有近4000人感染，其中22%患上了HUS。除了具有编码噬菌体的染色体整合志贺毒素2(Stx2)外，高毒力EHEC O104：H4还表达PAA质粒编码的聚集性黏附菌毛I(AAF/I；肠聚集性大肠杆菌的特征毒力特征)，这些特征介导其与培养的人上皮细胞紧密黏附。此外，EHEC O104：H4还表现出由接合pESBL质粒介导的超广谱β-内酰胺酶(ESBL)表型。我们的研究利用最先进的转录方法来进一步了解EHEC O104：H4的毒力和适合性决定因素。在第一个资助期，我们分析了PAA和pESBL质粒的初级转录本，并使用差异RNA序列进一步了解了它们的基因表达，这是一种定位转录起始点和非编码RNA的强大方法。此外，利用比较RNA-seq，我们鉴定了EHEC O104：H4与其他致病性较低和共生菌株之间共有的特征，但在相同条件下差异表达。我们推测，这种差异基因表达的情况可能对EHEC O104：H4的毒力和适合性有显著贡献。我们的转录组分析显示，在模拟肠道的条件下，与我们分析中包括的对照菌株相比，EHEC O104：H4中的中枢代谢基因下调。进一步的研究表明，用EHEC O104：H4的Stx2噬菌体裂解大肠杆菌K-12 MG1655后，其转录组和表型也发生了类似的变化。因此，在第二个资助期，我们将分析Stx2噬菌体携带对EHEC O104：H4宿主基因表达的影响。此外，我们还将研究Stx2噬菌体依赖转录组对EHEC O104：H4毒力和适合度的影响，并确定介导这种影响的噬菌体编码因子。最后但同样重要的是，我们将评估几个基因的生物学功能，这些基因在我们的分析中发现在EHEC O104：H4中上调，以前与大肠杆菌的毒力和适合度有关。我们预计，我们的结果将大大有助于我们对EHEC致病性的总体了解，特别是有助于对新出现的STX杂交毒株的风险评估。此外，识别与EHEC毒力相关的常见但不同调控的大肠杆菌决定因素将有助于病原菌的表型特征和监测，并最终治疗和/或预防该病，这将与公共卫生相关。

Enterohemorrhagic Escherichia coli (EHEC) can cause severe foodborne illness in humans, which is typically characterized by bloody diarrhea and progresses to hemolytic uremic syndrome (HUS) in circa 10% of the cases. EHEC O104:H4 was identified as the causative agent of the largest German outbreak (May-July 2011) during which nearly 4000 people were infected, and of them 22% developed HUS. Besides having a chromosomally integrated Shiga toxin 2 (Stx2) encoding phage, the highly virulent EHEC O104:H4 expresses pAA plasmid-encoded aggregative adherence fimbriae I (AAF/I; a characteristic virulence feature of enteroaggregative E. coli), which are mediating its tight adherence to cultured human epithelial cells. In addition, EHEC O104:H4 displays an extended spectrum beta-lactamase (ESBL) phenotype mediated by the conjugative pESBL plasmid.Our research exploits state of the art transcriptomic approaches to gain further insight into the virulence and fitness determinants of EHEC O104:H4. In the first funding period, we analyzed the primary transcriptomes of the pAA and pESBL plasmids and gained further insights into their gene expression using differential RNA seq, which is a powerful method for mapping of transcription start sites and non-coding RNAs. In addition, using comparative RNA-seq we identified features which are shared between EHEC O104:H4 and other less pathogenic and commensal strains, but are differentially expressed under identical conditions. We hypothesized that such cases of differential gene expression may significantly contribute to EHEC O104:H4 virulence and fitness. Our transcriptome analysis revealed that under conditions simulating the gut central metabolic genes are downregulated in EHEC O104:H4 in comparison to the control strains included in our analysis. Further investigations revealed that lysogenizing E. coli K-12 MG1655 with the Stx2 phage of EHEC O104:H4 resulted in analogous changes in the transcriptome and phenotype. Therefore, in the second funding period we will analyze the impact of Stx2 phage carriage on EHEC O104:H4 host gene expression. Moreover, we will investigate the impact of Stx2 phage-dependent transcriptome on EHEC O104:H4 virulence and fitness and identify the phage-encoded factors mediating it. Last but not least, we will evaluate the biological function of several genes, which were found upregulated in EHEC O104:H4 in our analysis and were previously linked to E. coli virulence and fitness. We anticipate that our results will significantly contribute to our general understanding of EHEC pathogenicity and in particular be beneficial for risk assessment of emerging Stx hybrid strains. Moreover, the identification of common but differentially regulated E. coli determinants associated with EHEC virulence will be of relevance to public health by facilitating phenotypic characterization and surveillance of pathogenic strains and eventually therapy and/or prevention of the disease.