Vessie

La vessie est l'organe du système urinaire dont la fonction est de recevoir l'urine terminale produite par les reins puis de la conserver avant son évacuation au cours de la miction. L'urine arrive dans la vessie par les uretères et est émise via l'urètre. Anatomie et physiologie La vessie est un organe musculaire pelvien creux de la forme d'une pyramide dont l'apex est étirée vers le haut et l'avant. Le point le plus déclive de la vessie est l'urètre par lequel l'urine sera évacuée lors de la miction. Ses parois sont constituées d'un muscle lisse, détrusor. Il est recouvert sur sa face intérieure d'un épithélium transitionnel (stratifié), l'urothélium. Le remplissage maximal habituel de la vessie est d'une capacité physiologique de 300 à 600 ml chez l'adulte. Au-delà, son remplissage est pathologique et la vessie peut acquérir une forme de globe, d'où le nom de globe vésical (elle peut contenir jusqu'a six litre avant rupture). La sensation de besoin se fait ressentir

Microtissue models for studying the dynamics of host-pathogen interactions in the urinary bladder

Kunal Sharma

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 âsolitaryâ 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

EPFL2021

Identification of genes required for host-cell adhesion and antibiotic persistence of uropathogenic Escherichia coli via high-content screening

Thomas Marie Simonet

Uropathogenic Escherichia coli (UPEC) are the most common bacterial pathogens causing urinary tract infections (UTIs). Driven by the development of antibiotic resistant UPEC strains, UTIs have become a major public health issue and generate substantial healthcare costs. Even in the absence of antibiotic resistance, UTIs are notoriously difficult to treat and have high rates of recurrence, mostly due to: 1. the ability of UPEC to adhere to and invade bladder epithelial cells, which protects them from the action of antimicrobial agents and the immune system 2. the presence in antibiotic-susceptible UPEC populations of a small fraction of so-called persister cells, which are killed at a slower rate than the bulk of the population and can therefore survive prolonged antibiotic exposureThese two features allow some bacteria to remain viable in a protected niche for the whole duration of the antibiotic treatment, potentially leading to infection relapse when the treatment is terminated.High-content screening (HCS), which uses microscopy as a screening tool, allows the visualization of bacterial phenotypes at the single-cell level and can therefore provide a powerful and unbiased tool to understand the genetic basis of these phenotypes. The present thesis describes two complementary high-content screening approaches aimed at identifying UPEC mutants with altered adhesion to bladder cells and with altered antibiotic persistence respectively.In a first chapter, we report preparatory steps that led to the construction of a transposon insertion library of UPEC mutants that is suitable for high-content imaging, using the CFT073 clinical UTI isolate as background strain.In the second chapter, we performed fluorescence microscopy-based high-content screening to identify mutants with defects in early adhesion to bladder epithelial cells. We recovered 82 mutants with decreased adhesion and 54 mutants with increased adhesion. Unexpectedly, nine low-adhesion ÂÂhitsÂÂ mapped to the two P pili operons encoded by CFT073, which are thought to mediate adhesion to kidney cells rather than bladder cells. Additionally, six high-adhesion hits mapped to the operon coding for F1C pili, and we show that this phenotype is linked to increased P pili synthesis. These results therefore reveal a critical role for P pili in UPEC adhesion to bladder epithelial cells, which may inform the development of novel anti-adhesion therapies to prevent UTI recurrence.In the third chapter, we combined microfluidics with time-lapse microscopy to identify mutants with altered persistence to fosfomycin (FOS) âÂÂ a cell-wall inhibitor used as first-line agent in the treatment of UTIs âÂÂ in synthetic human urine (SHU), which mimics the physiology of UPEC in real urine. We recovered four mutants with decreased persistence, three of which had defects in lipopolysaccharide (LPS) synthesis. These results identify the outer membrane of UPEC as a key component for survival to FOS treatment, thus opening up new avenues in the fight against antibiotic persistence.

EPFL2022

Identification of genes required for host-cell adhesion and antibiotic persistence of uropathogenic Escherichia coli via high-content screening

Thomas Marie Simonet

EPFL2022

Microtissue models for studying the dynamics of host-pathogen interactions in the urinary bladder

Kunal Sharma

EPFL2021

Microtissue models for studying the dynamics of host-pathogen interactions in the urinary bladder

Identification of genes required for host-cell adhesion and antibiotic persistence of uropathogenic Escherichia coli via high-content screening

Early invasion of the bladder wall by solitary bacteria protects UPEC from antibiotics and neutrophil swarms in an organoid model

Identification of genes required for host-cell adhesion and antibiotic persistence of uropathogenic Escherichia coli via high-content screening

Early invasion of the bladder wall by solitary bacteria protects UPEC from antibiotics and neutrophil swarms in an organoid model

Microtissue models for studying the dynamics of host-pathogen interactions in the urinary bladder