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.
Highlights
Clostridioides difficile (C. difficile), a gram positive, spore forming anaerobic bacterium, is a major cause of nosocomial diarrhea (Zhu et al, 2018)
Given that TcdA had previously been shown to inhibit Wnt/βcatenin signaling in the intestinal epithelial cells (IECs)-6 cells (Lima et al, 2014), we asked whether Wnt/β-catenin signaling could be inhibited in a mouse model of TcdA-induced ileitis, since epithelial cells interact with other cells in vivo
Because TcdA glucosylates Rho guanosine triphosphatases (GTPases), such as Rac1, which induces the recruitment of β-catenin to the nucleus (Wu et al, 2008; Pethe et al, 2011; Jamieson et al, 2015), we investigated whether upregulation of Rac1 through transfection of pcDNA3-EGFP-Rac1-Q61L could recover Wnt3a-induced β-catenin nuclear translocation in the intestinal epithelial (IEC-6) cells challenged with TcdA
Summary
Clostridioides difficile (C. difficile), a gram positive, spore forming anaerobic bacterium, is a major cause of nosocomial diarrhea (Zhu et al, 2018). Twenty mammalian genes encoding Rho GTPases have been identified of which Rac, Cdc, and RhoA are the prototypes and the best characterized (Jaffe and Hall, 2005). These signaling proteins regulate all the actin-dependent process like cell migration, phagocytosis, and cell contraction. Most Rho GTPases cycle between a GTP-bound active conformation and a GDP-bound inactive conformation (Wennerberg and Der, 2004), acting as molecular switches Not surprisingly, this family of molecules plays central roles in maintenance of health, and their dysregulation often results in disease. Anomalous signaling of Rho GTPases is found in many human cancers and may be attributed to several mechanisms, such as overexpression of Rho GTPases with oncogenic activity or alterations of upstream regulators or downstream effectors (Vega and Ridley, 2008)
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