Abstract

Urinary tract infections (UTIs) are amongst the most common bacterial infections and are the second-most frequent reason for antibiotic prescriptions. Moreover, in about 25% of all treated cases, recurrence of infection occurs. Uropathogenic Escherichia coli (UPEC) is the most common causative agent of UTIs. Much of our current understanding about UTIs has come from mouse models of UTI which have highlighted the intracellular lifestyle of this pathogen in the forms of intracellular bacterial communities (IBCs) as the prominent cause for bacterial persistence. Recurrent infections are thought to be caused by bacterial fluxing from persistent IBCs in the umbrella cell layer of the epithelia in the bladder. Although in vitro model systems of UTIs have been developed to study acute phases of infection including the formation of IBC and their dispersal, they suffer from certain shortcomings. For example, these models lack vasculature and the possibility to impose mechanical stresses, such as those experienced by the cells during bladder filling and voiding. Moreover, the design of these in vitro systems makes it difficult to study long-term UPEC persistence when exposed to antibiotic or neutrophil-mediated stresses. In the last two decades, two bioengineering approaches have emerged to generate functional and physiological tissues: organoid and organ-chip systems. Differentiating cells self-organize in a hydrogel and enable the formation of organoids, which mimic various aspects of organ physiology and function in a three-dimensional structure. Organ-chip systems rely upon co-culturing pre-differentiated cells across a synthetic membrane in a defined geometry. The organ-chip systems allow dynamic control over environmental (nutritive) state and over the mechanical stresses experienced by the emulated tissue. In this thesis, I showcase the development of model systems to study UPEC pathogenesis using both these approaches. In the first part of this thesis, we developed a bladder organoid model of UPEC infection that recapitulates the stratified bladder architecture within a volume suitable for high-resolution live-cell imaging. Bacteria injected into the organoid lumen rapidly enter umbrella cells and proliferate to form IBCs. Unexpectedly, we identified populations of individual “solitary” bacteria that form independently of IBCs and which penetrate deeper layers of the organoid wall, and evade killing by antibiotics and neutrophils. Volumetric electron microscopy revealed that these solitary bacteria may be intracellular or pericellular (sandwiched between uroepithelial cells) and are rod-shaped and flagellated, unlike coccoid-shaped and unflagellated bacteria within IBCs. Bacterial fluxing from IBCs is therefore not an essential requirement for the establishment of persistent bacterial populations in the bladder wall. The dynamic responses of IBCs to host stresses and antibiotic therapy are difficult to assess in situ. In another study, we focussed on examining the lifecycle and persistence of IBCs during early stages of UTIs. We developed a human bladder-chip model wherein superficial umbrella cells and bladder microvascular endothelial cells are co-cultured under flow in urine and nutritive media respectively. Bladder filling and voiding was mimicked mechanically by application and release of linear strain. Using time-lapse microscopy, we show that rapid recruitment of neutrophils from the vascular channel

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