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MIRAVA POLYSCOPE – All in one and on for all: the perfect image
Science beyond Barriers

abberior instruments

Cell Biology

2023
Toxins

A Colonic Organoid Model Challenged with the Large Toxins of Clostridioides difficile TcdA and TcdB Exhibit Deregulated Tight Junction Proteins

Authors:

Martina Schneemann, Lucas Heils, Verena Moos, Franziska Weiß, Susanne M. Krug, January Weiner, Dieter Beule, Ralf Gerhard, Jörg-Dieter Schulzke, Roland Bücker

Keywords:

intestinal epithelial human colonic organoid monolayer; claudin; leak flux diarrhea; leaky gut; super-resolution STED microscopy; tricellulin; channel-forming claudin-2; RNA-seq pathway analysis; cytokines; actin

Abstract:

Background: Clostridioides difficile toxins TcdA and TcdB are responsible for diarrhea and colitis. Lack of functional studies in organoid models of the gut prompted us to elucidate the toxin’s effects on epithelial barrier function and the molecular mechanisms for diarrhea and inflammation. Methods: Human adult colon organoids were cultured on membrane inserts. Tight junction (TJ) proteins and actin cytoskeleton were analyzed for expression via Western blotting and via confocal laser-scanning microscopy for subcellular localization. Results: Polarized intestinal organoid monolayers were established from stem cell-containing colon organoids to apply toxins from the apical side and to perform functional measurements in the organoid model. The toxins caused a reduction in transepithelial electrical resistance in human colonic organoid monolayers with sublethal concentrations. Concomitantly, we detected increased paracellular permeability fluorescein and FITC-dextran-4000. Human colonic organoid monolayers exposed to the toxins exhibited redistribution of barrier-forming TJ proteins claudin-1, -4 and tricellulin, whereas channel-forming claudin-2 expression was increased. Perijunctional F-actin cytoskeleton organization was affected. Conclusions: Adult stem cell-derived human colonic organoid monolayers were applicable as a colon infection model for electrophysiological measurements. The TJ changes noted can explain the epithelial barrier dysfunction and diarrhea in patients, as well as increased entry of luminal antigens triggering inflammation.

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