Paper Title
Clostridioides Difficile Flagellin Activates the NLRC4 in Flamma Some Pathway

Abstract
Clostridioides difficile (C. difficile), an anaerobic bacterium, is a major cause of intestinalinfection, nosocomial diarrhea, and colitis. Recently, we showed that C. difficile flagellin(FliC) contributes with toxins to the gut damage. Indeed, FliC interacts with the innateimmune receptor TLR5 to activate NF-kB and MAPK signaling pathways, leading to a pro-inflammatory response. Otherwise, Salmonella flagellin is known to activate theinflammasome signaling pathways, particularly the NAIP-NLRC4 inflammasome.C. difficile is an extracellular bacterium, but for C. difficileFliC to possibly interact withintracellular proteins of the inflammasome, FliC would have to be able to enter epithelialcells. Our first aim was to detect the internalization of FliC; we thus produced recombinantGFP-FliC using the Golden Gate technique. Confocal microscopy assays allowed us to detectFliC internalization within 4 hours in intestinal Caco-2/TC7 epithelial cells. In addition,colocalization between GFP-FliC and pro-caspase-1was observed, suggesting the NLRC4inflammasome formation through the complexation of the NLRC4 proteins, pro-caspase-1and FliC. Western blotting and qPCR experiments were performed to study the activation ofNLRC4 by recombinant FliC in Caco-2/TC7 epithelial cells. Results showed an increase ofNLRC4 phosphorylation in FliC stimulated cells compared to cells stimulated by arecombinant truncated GFP-FliC (lacking the C-terminal region which recognizes themolecules of the inflammasome). We also observed cleavage of pro-caspase-1 into twosubunits, p20 and p10, 6 h after stimulation by FliC. The p20 release with the Gasdermincleavage strongly suggests the caspase-1 and NLRC4 inflammasome activation. Similarly,overexpression of the interleukins (IL-1β, IL-18, and IL-33) encoding genes was detectedafter cell stimulation, regulated by the NF-κβ signaling pathway. Altogether, our resultssuggest that FliC internalizes into the TC7 cells and plays a role in the inflammatory processby stimulating not only TLR5 but also the intracellular inflammatory pathway involvinginflammasomes. Targeting the internalization of FliC may be a possible potential therapeuticstrategy in the future. Keywords - Clostridioides difficile; Flagellin; intestinal epithelial cells; inflammasome signaling pathway;NLRC4; pro-caspase-1; Gasdermin; pro-inflammatory cytokines.