Influence of environmental cues on virulence factor regulation of Enterohemorrhagic Escherichia coli

Armansperg, Benedikt von (2024): Influence of environmental cues on virulence factor regulation of Enterohemorrhagic Escherichia coli. Dissertation, LMU München: Medizinische Fakultät

Abstract

Enterohemorrhagic Escherichia coli (EHEC) is a major food-borne human pathogen causing severe gastrointestinal disease, particularly acute diarrhoea, and haemorrhagic colitis, with an elevated risk in children and elderly. Both age groups have a higher risk to develop severe complications, including the development of haemolytic uremic syndrome (HUS), a life-threatening condition closely associated with the production of Shiga toxin 2 (Stx2) by the pathogen. Certain host factors, including age, gender and an elevated immune response towards Stx2, have been associated with an increased risk of HUS development. Likewise, Stx2 production is promoted by the induction of bacterial stress response like the SOS response mediated via RecA. Therefore, the use of certain antibiotics as therapy for EHEC infection are contraindicated. Furthermore, other environmental risk factors for the development of HUS are poorly characterized. The aim of this thesis was to investigate the role of environmental factors including human-targeted drugs and the microbiome in influencing the expression of EHEC virulence genes, particularly Stx2 production. To address these aims, I established a high-throughput assay to systematically analyse the impact of various compounds on stx2 expression. Utilizing an E. coli C600-based luciferase reporter strain (CWgluc), I optimized the assay for scalability and reproducibility. A range of human-targeted drugs has been shown to have antibacterial activity. Hence, I hypothesized that drugs may also influence stx2 expression. I used the established assay to screen a comprehensive chemical library of Food and Drug Administration (FDA) and European Medicines Agency (EMA)-approved human-targeted drugs for their effects on stx2 expression. Several compounds with either stimulatory or inhibitory effects on Stx2 production were identified and confirmed through several validation processes. Notably, certain antibiotic classes like quinolones and fluoroquinolones were highly represented among stx2 inducing compounds, as well as beta-lactam antibiotics, belonging to the cephalosporin and penicillin cluster. Chemotherapeutic compounds evoked a significant inducing effect on stx2, especially the compounds dacarbazine and streptozotocin. Additionally, acetylsalicylic acid (aspirin), a widely taken medication, showed clear signs of stx2 inducing effects. On the other hand, several compounds showed inhibitory effects on stx2 expression. Especially antibiotics inhibiting the protein biosynthesis like azithromycin or clarithromycin had a strong inhibitory effect. But also, non-antibiotic compounds like levodopa showed inhibition of stx2 expression. Overall, this screen identified both, stx2 inducing and inhibiting compounds, pointing at potential risk factors to develop severe disease on the one hand, and possible therapeutic avenues for mitigating HUS development on the other. Additionally, I analysed microbiome datasets from faecal samples of patients obtained during the haemolytic uremic syndrome associated E. coli (HUSEC) outbreak in Northern Germany in 2011 to identify microbiome-based risk factors indicating disease severity. The analysis revealed distinct microbiota signatures associated with HUS, pointing at a potential role of specific bacterial taxa in modulating EHEC virulence or disease outcome. In particular, the presence of bacteria encoding the genotoxin colibactin (pks island), which was previously shown to induce stx2 expression in vitro, was elevated within the HUS patient group. These data may point at a potential mechanism which increases the patient risk to develop HUS. In conclusion, this thesis provides significant insights into the interplay of environmental cues, the intestinal microbiota, and EHEC pathogenesis. The established high-throughput assay to quantify Stx2 production in live bacteria offers a valuable tool for further research into Stx2 regulation and the identification of potential drug-based interventions. The findings underscore the importance of considering microbiome composition and drug interactions in managing EHEC infections and preventing HUS. This holistic approach could pave the way for more effective and personalized treatments, ultimately improving patient outcomes in the fight against EHEC-related diseases.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Stx, EHEC, Shiga toxin, Prophage induction, Human targeted-drugs
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 17. Dezember 2024
• 1. Bericht­erstatter:in: Stecher-Letsch, Barbara