Host-pathogen interactions: dissecting the multifactorial virulence of Enteroaggregative Escherichia coli and exploring epithelial responses to bacterial infections

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen increasingly associated with persistent diarrhoea, especially in children and vulnerable populations. Its pathogenicity is multifactorial, involving strong adhesion to the intestinal mucosa, robust biofilm formation, and the secretion of toxins and modulators that trigger inflammation and epithelial responses. Its ability to persist in the intestinal niche, combined with the rise of antimicrobial resistance, highlights the urgent need for new therapeutic approaches beyond conventional antibiotics. This thesis investigates EAEC pathogenesis by identifying key bacterial determinants involved in colonisation and persistence, and it explores host epithelial responses to bacterial infections using advanced cellular models. First, we characterised the AcrAB-TolC efflux pump, traditionally known for its role in multidrug resistance, demonstrating its key contribution to EAEC virulence. Loss of AcrB compromised key virulence-associated traits, including biofilm formation, extracellular DNA production, and the characteristic aggregative adherence phenotype, and led to attenuated virulence in vivo in Caenorhabditis elegans. Second, we analysed AggR, an AraC/XylS-family transcriptional regulator that orchestrates multiple virulence pathways, including the expression of aggA, which encodes the major structural subunit of aggregative adherence fimbriae (AAF). We demonstrate that medium-chain fatty acids (caprylic, lauric and decenoic) strongly inhibit EAEC pathogenicity by downregulating aggR and aggA expression and interfering with AggR activity, resulting in reduced biofilm formation and epithelial adhesion. Finally, we established a human colon organoid model to examine epithelial responses to infection. Using an ExPEC strain as proof-of-concept, we showed that different bacterial loads induce distinct epithelial outcomes: low infection levels promote adaptive transcriptional reprogramming, including metaplastic gene expression, whereas high loads trigger strong inflammatory responses. Altogether, these findings clarify key aspects of EAEC virulence and its modulation by host and environmental factors, suggesting new avenues to limit colonisation and persistence through novel therapeutic interventions.