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

Lara Petersen,Svenja Stroh,Klemens Rottner,Guntram A. Grassl,Harald Genth,Cord Brakebusch,Jörg Fahrer,Dennis Schöttelndreier

doi:10.3389/fmicb.2022.846215

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.

Highlights

The Clostridioides difficile Toxin B (TcdB, 270 kDa) [together with Toxin A (TcdA, 307 kDa)] is causative for the pathology of C. difficile infection (CDI), ranging from mild diarrhea to pseudomembranous colitis (PMC; Smits et al, 2016)
SV40-immortalized Rac1fl/fl mouse embryonic fibroblasts (MEFs) that expressed Cyclin D1, Cyclin D2, and Cyclin D3 served as a positive control (Supplementary Figure S1)
In TcdB-treated Human intestinal organoids (HIOs), inhibition of Rac/Cdc42-PAK signaling by TcdB, coincided with suppression of Cyclin D1, with the kinetics of Cyclin D1 suppression being almost comparable to that of Rac/Cdc42 glucosylation (Figures 1A,B)

Summary

Introduction

The Clostridioides difficile Toxin B (TcdB, 270 kDa) [together with Toxin A (TcdA, 307 kDa)] is causative for the pathology of C. difficile infection (CDI), ranging from mild diarrhea to pseudomembranous colitis (PMC; Smits et al, 2016). Upon internalization into the cytosol, the glucosyltransferase domain catalyzes divalent metal ion-dependent mono-O-glucosylation and thereby inactivation of small GTPases of Rho and Ras families (Genth et al, 2014, 2016, 2018). While TcdB glucosylates GTPases of the Rho, Rac, and Cdc subfamilies, the related TcdA exhibits a broader substrate profile, as it glucosylates Ras subtype GTPases in addition to Rho/Rac/Cdc subtype GTPases (Genth et al, 2018). Upon treatment of cultured cells with TcdB, glucosylation of Rho GTPases results in actin depolymerization (Mitchell et al, 1987; May et al, 2013) and cell death including upregulation of the cell death-regulating GTPase RhoB (Huelsenbeck et al, 2007; Matarrese et al, 2007; Farrow et al, 2013; Wohlan et al, 2014)

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