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Investigating Intracellular Mechanisms of Infection Stone Formation

Applicant Dr. Roman Herout
Subject Area Reproductive Medicine, Urology
Term from 2020 to 2023
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 447437311
 
Final Report Year 2023

Final Report Abstract

Proteus mirabilis is a Gram-negative, rod-shaped bacterium that can cause infections in humans. Proteus is an avid biofilm former and possesses the enzyme urease, with which the pH can be raised by splitting urea. This pH elevation can lead to the precipitation of calcium ammonium phosphate (struvite). The exact processes by which Proteus mirabilis attaches to and invades into kidney and bladder cells and the role that bacterial surface components (BSC) play in these processes remain unknown. We conducted experiments in which the adhesion to catheters and the adhesion to and invasion into kidney and bladder cells were studied. We used the wildtype as well as seven different mutant strands of the bacterium. In these mutant strands, genes had been altered that encoded for proteins that play a role in adhesion and invasion processes. The aim was that these mutant strands no longer produce functional adhesion and invasion proteins so that their influence on these processes could be studied in detail. Two of these genes were reinserted using a plasmid (complemented strands). In vitro, catheter adhesion, adhesion to and invasion into two different renal cell lines (HEK293 and A498) and a bladder cell line (T24) were investigated. Overall, mutant strands’ adhesion to catheters and the different cell types tested was significantly reduced compared to the wildtype, while no invasion of cells was evident at 24h. We then carried out in vivo experiments in an established mouse model of catheter-associated urinary tract infections (CAUTI). For this purpose, a 4 mm long catheter piece (the tip of an angiocatheter) was inserted percutaneously into the urinary bladder of mice under ultrasound control and subsequently Proteus mirabilis was introduced percutaneously into the bladder. On the fourth day of the experiment, urine was collected from the mice percutaneously and aseptically under ultrasound control, and the mice were then euthanized. PM WT showed a greater number of planktonic (urine) bacteria, bacteria adherent to catheters, and bacteria adherent to/invading into bladder tissue when compared to the mutant strands. The complementation of mutated BSC genes resulting in the biggest defects restored the invasion phenotype both in vitro and in vivo. BSCs play a critical role at various steps in the pathogenicity of Proteus mirabilis including adhesion to indwelling medical devices and adhesion/invasion of urinary tissue in vivo.

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