Clostridioides difficile Toxin A-Induced Wnt/β-Catenin Pathway Inhibition Is Mediated by Rac1 Glucosylation

Abstract

Clostridioides difficile toxin A (TcdA) has been shown to inhibit cellular Wnt signaling, the major driving force behind the proliferation of epithelial cells in colonic crypts, likely through the inhibition of β-catenin nuclear translocation. Herein, we aimed to advance the understanding of this mechanism by replicating the findings in vivo and by investigating the specific role of Rac1, a member of the Rho GTPase family, on the inhibition of the Wnt-induced β-catenin nuclear translocation triggered by TcdA. To investigate the effects of TcdA on the Wnt/β-catenin pathway in vivo , we injected the ileal loops of C57BL/6 mice with TcdA [phosphate-buffered saline (PBS) as the control] to induce C. difficile disease-like ileitis. After 4 h post-injection, we obtained ileum tissue samples to assess Wnt signaling activation and cell proliferation through Western blotting, immunohistochemistry, and qPCR. To assess the role of Rac1 on Wnt signaling inhibition by TcdA, we transfected rat intestinal epithelial cells (IEC-6) with either a constitutively active Rac1 plasmid (pcDNA3-EGFP-Rac1-Q61L) or an empty vector, which served as the control. We incubated these cells with Wnt3a-conditioned medium (Wnt3a-CM) to induce Wnt/β-catenin pathway activation, and then challenged the cells with TcdA. We assessed Wnt signaling activation in vitro with TOP/FOPflash luciferase assays, determined nuclear β-catenin translocation by immunofluorescence, measured cyclin D1 protein expression by Western blotting, and quantified cell proliferation by Ki67 immunostaining. In vivo , TcdA decreased β-catenin, cyclin D1, and cMYC expression and inhibited the translocation of β-catenin into the nucleus in the ileum epithelial cells. In addition, TcdA suppressed cell proliferation and increased Wnt3a expression, but did not alter Rac1 gene expression in the ileum tissue. In vitro , constitutively active Rac1 prevented Wnt signaling inhibition by enabling the β-catenin nuclear translocation that had been blocked by TcdA. Our results show that TcdA inhibits Wnt/β-catenin pathway in vivo and demonstrate that this inhibition is likely caused by a Rac1-mediated mechanism.

The Essential Role of Rac1 Glucosylation in Clostridioides difficile Toxin B-Induced Arrest of G1-S Transition

“…Furthermore, the related TcdA has been shown to suppress Cyclin D1 expression ( Bezerra Lima et al, 2014 ). Interestingly, TcdA-induced inhibition of the Wnt/β-Catenin Pathway is shown to be preserved upon expression of non-glucosylatable Rac1-Q61L ( Martins et al, 2020 ). In sum, Rac1 seems to be the critical upstream regulator of G1 phase Cyclins on the level of Rho GTPases, which glucosylation by either TcdA or TcdB results in Cyclin D1 suppression.…”

Clostridioides difficile Toxins: Host Cell Interactions and Their Role in Disease Pathogenesis

“…The signs and symptoms following CDI are influenced by various virulence factors, including the production of toxins and surface proteins [ 3 , 9 , 24 ]. The pathogenesis is primarily driven by the activity of toxins A and B, encoded in the pathogenicity locus of the C. difficile genome [ 25 , 26 ].…”

Clostridioides difficile Toxin B Induced Senescence: A New Pathologic Player for Colorectal Cancer?

“…To date, there is no in vivo demonstration of cellular senescence during CDI, although studies on the effects of Tcds in the tissue of mice during CDI demonstrated changes in the expression of cell cycle inhibitors and CDKIs indicative of cell cycle arrest [24,25], strongly suggesting that cellular senescence could occur during CDI in vivo.…”