Bacterial and host determinants of uropathogenic E. coli virulence in the human urothelial environment

Urinary tract infections (UTIs) are a pressing issue affecting ~400 million people worldwide each year. In the UK alone, ~6 million antibiotic prescriptions are written annually for UTIs. However, their effectiveness is threatened due to increasing antimicrobial resistance (AMR), already contributing to ~260K UTI-associated deaths globally. Additionally, UTIs have a high recurrence rate (25-30%), requiring repeated antibiotic courses which further exacerbate the AMR crisis. To address this challenge, development of new UTI therapies is crucial, for which understanding the underlying mechanisms of disease is key. Most UTIs start in the bladder and can escalate to more severe conditions if left untreated. Both the body's response to the infection and the characteristics of the bacteria involved play important roles in how UTIs develop and progress. The main bacteria responsible for UTIs are uropathogenic E. coli (UPEC), accounting for ~80% of all community-acquired UTIs. However, the relationship between bacterial virulence, how the body responds, and the severity of the infection is not fully understood. One obstacle to studying UTIs is the limitations of research models. While mice have been valuable, their bladder environment differs from humans in several important ways. Other methods, like using human cell lines or patient-derived cells, also have drawbacks. To address these limitations, we have recently developed a radically new microtissue model called 3D urine-tolerant human urothelium (3D-UHU), which mimics the human urothelium more accurately. This model is unique and allows us, for the first time, to study how E. coli and the human urothelium interact in a tightly controlled environment, providing mechanistic insights difficult to achieve with other models. Our initial published experiments with E. coli-infected 3D-UHU showed a variety of host-pathogen interactions, including invasion, intracellular growth, bacterial expulsion, biofilm formation, barrier disruption, urothelial cell shedding, cytopathicity and cytokine secretion. Notably, UPEC strains provoked different responses compared with non-pathogenic strains derived from the urinary microbiota. In this project, we aim to uncover how E. coli virulence factors affect human urothelial physiology, and in turn how the changes induced correspond to UTI disease severity. Our objectives include (1) using a comprehensive mutant library to create a broad linkage map of UPEC virulence factors in the human cell setting; (2) understanding how pathogen and host respond when they interact at the urothelium interface using a combined bacterial-human transcriptomic approach; and (3) defining the mechanistic interplay between bacterial virulence factor expression in patient-derived E coli isolates, urothelial responses and clinical severity. Our research is both exciting and timely, leveraging the new availability of the 3D-UHU model, cutting-edge tools to dissect E. coli and urothelial biology, and access to a wide range of E. coli clinical isolates from well-characterised patients. Our project will shed light on UTIs in a human context, uncovering new insights into how bacteria cause infections and how the body responds. Overall, our work has potential for wide impact. Despite being a common problem, UTI research – like many diseases that primarily affect women – has been historically overlooked. By understanding the mechanisms of disease, we hope to develop new drugs that target critical bacterial processes that damage the urothelium, alongside host-directed drugs that dampen problematic urothelial responses. Given the increasing AMR threat, our work has the potential to improve not only outcomes for individuals with UTIs, but more broadly, the treatment of other bacterial infections as well.