Ramirez Finn, L. | bioRxiv Science

bioRxiv2024

A simple ex vivo bladder infection model permits study of host-pathogen interactions in UTI

Urinary tract infections (UTI) are one of the most common infections, worldwide. To understand mechanisms of UTI pathogenesis and find new treatments, researchers often use animal models, such as mice or rats. However, studying certain phenotypes in animals can be difficult. Additionally, using animals in research comes with significant administrative and ethical challenges. To address these challenges, we developed a simple, reproducible, and cost-effective model to study UTI using donated mouse bladder tissue that would otherwise be discarded. This model allows researchers of all experience levels to study interactions between the host and pathogen in a controlled environment. We tested uropathogenic E. coli colonization and invasion of isolated urothelial sheets from 30 minutes to 24 hours, finding that bacterial burden in our ex vivo model was comparable to in vivo UTI mouse models. To optimize reproducibility, we tested multiple variables, including technical parameters, such as incubator conditions, and biological factors, such as biological sex or prior pregnancy in the donor mouse. This method offers several advantages, including assessment of early host-pathogen interactions, immune cell uptake of bacteria, the impact of age and sex of donor animals in infection, and diverse bacterial strains, mutants, or treatments. In addition, in some countries, sharing material recovered from animals sacrificed for other reasons does not require additional ethical approval by the receiving laboratory, providing a resource for labs without access to animals and reducing administrative burden. Given the breadth of the model with respect to sex, age, mouse and bacterial strain, and the ability to test any parameter that can be included in a 96-well plate, we believe this model will be useful to UTI researchers, with potential application beyond infection or even beyond the bladder to other tissues.

immunology↗

bioRxiv2024

Bacterial growth under confinement requires transcriptional adaptation to resist metabolite-induced turgor pressure build-up

Bacterial proliferation often occurs in confined spaces, during biofilm formation, within host cells, or in specific niches during infection, creating mechanical constraints. We investigated how spatial confinement and growth-induced mechanical pressure affect bacterial physiology. Here, we found that, when proliferating in a confining microfluidic-based device with access to nutrients, Escherichia coli cells generate forces in the hundreds of kPa range. This pressure decouples growth and division, producing shorter bacteria with higher protein concentrations. This leads to cytoplasmic crowding, which ultimately arrests division and stalls protein synthesis. In this arrested state, the pressure produced by bacteria keeps increasing. A minimal theoretical model of bacterial growth predicts this novel regime of steady pressure increase in the absence of protein production, that we named overpressurization. In this regime, the Rcs pathway is activated and that abnormal shapes appear in rcs mutant populations only when they reach the overpressurized state. A uropathogenic strain of E. coli displayed the same confined growth phenotypes in vitro and requirement for Rcs in a mice model of urinary tract infection, suggesting that these pressurized regimes are relevant to understand the physiopathology of bacterial infections.

microbiology↗