Clostridioides difficile Flagellin Activates the Intracellular NLRC4 Inflammasome

Abstract

Clostridioides difficile (C. difficile), is a major cause of nosocomial diarrhea and colitis. C. difficile flagellin FliC contributes toxins to gut inflammation by interacting with the immune Toll-like receptor 5 (TLR5) to activate nuclear factor-kappa B (NF-kB) and mitogen-activated protein kinase (MAPK) signaling pathways. Flagella of intracellular pathogens can activate the NLR family CARD domain-containing protein 4 (NLRC4) inflammasome pathway. In this study, we assessed whether flagellin of the extracellular bacterium C. difficile internalizes into epithelial cells and activates the NLRC4 inflammasome. Confocal microscopy showed internalization of recombinant green fluorescent protein (GFP)-FliC into intestinal Caco-2/TC7 cell line. Full-length GFP-FliC activates NLRC4 in Caco-2/TC7 cells in contrast to truncated GFP-FliC lacking the C-terminal region recognized by the inflammasome. FliC induced cleavage of pro-caspase-1 into two subunits, p20 and p10 as well as gasdermin D (GSDMD), suggesting the caspase-1 and NLRC4 inflammasome activation. In addition, colocalization of GFP-FliC and pro-caspase-1 was observed, indicating the FliC-dependent NLRC4 inflammasome activation. Overexpression of the inflammasome-related interleukin (interleukin (IL)-1β, IL-18, and IL-33) encoding genes as well as increasing of the IL-18 synthesis was detected after cell stimulation. Inhibition of I-kappa-B kinase alpha (IKK-α) decreased the FliC-dependent inflammasome interleukin gene expression suggesting a role of the NF-κB pathway in regulating inflammasome. Altogether, these results suggest that FliC internalizes into the Caco-2/TC7 cells and activates the intracellular NLRC4 inflammasome thus contributing to the inflammatory process of C. difficile infection.

Flow-Induced Shear Stress Primes NLRP3 Inflammasome Activation in Macrophages via Piezo1

“…We anticipated that shear stress would only prime the NLRP3 inflammasome for activation and would not affect the priming of the NLRP1 and AIM2 inflammasomes since previous studies suggested that shear stress would not prime these inflammasomes. , While NF-kB is involved in the priming of the NLRC4 inflammasome pathway, Gram-negative bacteria are required to initiate activation; so, even if shear stress could prime this pathway for activation, the NLRC4 inflammasome could not become activated. − To confirm this, we separately primed NLRP3 KO and caspase-1 KO iBMDMs with 3 magnitudes of shear stress for 30 min, followed by 1 h nigericin treatment before imaging via confocal microscopy (Figure A,B). Similar to the WT iBMDMs used in this study, the KO iBMDMs were engineered to express a CFP tag on the ASC adaptor protein.…”

Clostridioides difficile Flagella

“…This was demonstrated by confocal microscopy with the observation of the internalization of FliC tagged with a green fluorescent protein (GFP) into the intestinal Caco-2/TC7 cell line. Further, the activation of NLRC4, the cleavage of pro-caspase-1 into two subunits, p20 and p10, and also the cleavage of gasdermin D, were shown, suggesting that the caspase-1 and NLRC4 inflammasome activation by FliC contributes to the inflammatory process of C. difficile infection [53].…”

Influences of bacterial extracellular vesicles on macrophage immune functions

“…Additionally, bacterial EVs containing flagellin can induce pyroptosis in macrophages by activating the NLRC4 inflammasome rather than the NLRP3 inflammasome ( Bitto et al., 2018 ). Unlike the NLRP3 inflammasome, the NLRC4 inflammasome specifically recognizes bacterial flagellin and flagellin-like molecules, leading to a more specific and rapid activation ( Gram et al., 2021 ; Chebly et al., 2022 ). For example, Salmonella EVs containing flagellin can quickly activate the NLRC4 inflammasome in a shorter time compared to flagellin-deficient E. coli and Salmonella EVs ( Yang et al., 2020 ).…”