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

Abstract

Clostridioides difficile infection (CDI) in humans causes pseudomembranous colitis (PMC), which is a severe pathology characterized by a loss of epithelial barrier function and massive colonic inflammation. PMC has been attributed to the action of two large protein toxins, Toxin A (TcdA) and Toxin B (TcdB). TcdA and TcdB mono-O-glucosylate and thereby inactivate a broad spectrum of Rho GTPases and (in the case of TcdA) also some Ras GTPases. Rho/Ras GTPases promote G1-S transition through the activation of components of the ERK, AKT, and WNT signaling pathways. With regard to CDI pathology, TcdB is regarded of being capable of inhibiting colonic stem cell proliferation and colonic regeneration, which is likely causative for PMC. In particular, it is still unclear, the glucosylation of which substrate Rho-GTPase is critical for TcdB-induced arrest of G1-S transition. Exploiting SV40-immortalized mouse embryonic fibroblasts (MEFs) with deleted Rho subtype GTPases, evidence is provided that Rac1 (not Cdc42) positively regulates Cyclin D1, an essential factor of G1-S transition. TcdB-catalyzed Rac1 glucosylation results in Cyclin D1 suppression and arrested G1-S transition in MEFs and in human colonic epithelial cells (HCEC), Remarkably, Rac1−/− MEFs are insensitive to TcdB-induced arrest of G1-S transition, suggesting that TcdB arrests G1-S transition in a Rac1 glucosylation-dependent manner. Human intestinal organoids (HIOs) specifically expressed Cyclin D1 (neither Cyclin D2 nor Cyclin D3), which expression was suppressed upon TcdB treatment. In sum, Cyclin D1 expression in colonic cells seems to be regulated by Rho GTPases (most likely Rac1) and in turn seems to be susceptible to TcdB-induced suppression. With regard to PMC, toxin-catalyzed Rac1 glucosylation and subsequent G1-S arrest of colonic stem cells seems to be causative for decreased repair capacity of the colonic epithelium and delayed epithelial renewal.

Neutralising Effects of Different Antibodies on Clostridioides difficile Toxins TcdA and TcdB in a Translational Approach

“…However, one should cautiously evaluate toxin effects in different species instead of extrapolating results from one species to another. Human organoid and/or intestinal epithelial systems have been used previously to assess the effects of TcdA and TcdB [ 45 , 46 , 53 ]. Using 2D and 3D approaches on canine intestinal organoids, we showed that toxin effects are not the same in small and large intestinal-derived organoids, even from the same species, so results from human studies may not be transferred directly to other organisms.…”

Applications of human organoids in the personalized treatment for digestive diseases

“… 237 , 238 TcdB of C. difficile infection of HIOs inhibits stem cell repair capacity and delays epithelial cell renewal. 249 Infection with the major foodborne enteric pathogen Listeria affects goblet and paneth cell numbers, upregulates muc2 and lyz expression, and reduces the expression of genes related to the Notch pathway. Increased secretion of proinflammatory cytokines disrupts organoid morphology.…”

“… 248 Clostridioides difficile Toxin B (TcdB) infection of HIOs inhibits stem cell repair capacity and delays epithelial cell renewal. 249 Colonization of Helicobacter pylori is associated with GC, which causes most gastric ulcers by inducing hypersecretion of gastric acid. 250 – 252 Gastric organoids from different sources have been used to replicate pathological conditions in vivo and to robustly mimic the gastric epithelial response to H. pylori infection in an in vitro system.…”